Systems and Methods for Nucleic Acid Expression In Vivo

ABSTRACT

The present invention provides compositions, systems, kits, and methods for generating expression of one or more proteins and/or biologically active nucleic acid molecules in a subject (e.g., at therapeutic levels for extended periods required to produce therapeutic effects). In certain embodiments, systems and kits are provided that comprise a first composition comprising a first amount of polycationic structures, and a second composition comprising a therapeutically effective amount of expression vectors (e.g., non-viral expression vectors not associated with liposomes) that are CpG-free or CpG-reduced, where the expression vectors comprise a first nucleic acid sequence encoding: i) a first therapeutic protein or proteins, and/or ii) a first biologically active nucleic acid molecule or molecules.

The present application is a divisional of U.S. patent application Ser.No. 15/268,000, filed Sep. 16, 2016, now allowed, which claims priorityto U.S. Provisional Application Ser. No. 62/220,646 filed Sep. 18, 2015,each of which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention provides compositions, systems, kits, and methodsfor expression of one or more proteins or biologically active nucleicacid molecules in a subject, human or non-human mammal, (e.g., attherapeutic levels for the extended periods of time required to producetherapeutic effects). In certain embodiments, systems and kits areprovided that comprise a first composition comprising polycationicstructures (e.g., empty cationic liposomes, cationic micelles, cationicemulsions, or cationic polymers) and a second composition comprisingexpression vectors (e.g., non-viral expression vectors not associatedwith liposomes or other carriers) that are CpG-free or CpG-reduced, thatcomprise a first nucleic acid sequence encoding: i) a first therapeuticprotein or proteins, and/or ii) a first biologically active nucleic acidmolecule or molecules. In other embodiments, such first and secondcompositions are sequentially administered (e.g., systemically) to asubject such that the therapeutic protein and/or the biologically activenucleic acid molecule is/are expressed in the subject (e.g., at atherapeutic level, for at least 5 or at least 50 days, such that adisease or condition is treated or a physiological or disease trait isaltered).

BACKGROUND

The simplest non-viral gene delivery system uses naked expression vectorDNA. Direct injection of free DNA into certain tissues, particularlymuscle, has been shown to produce high levels of gene expression, andthe simplicity of this approach has led to its adoption in a number ofclinical protocols. In particular, this approach has been applied to thegene therapy of cancer where the DNA can be injected either directlyinto the tumor or can be injected into muscle cells in order to expresstumor antigens that might function as a cancer vaccine.

Although direct injection of plasmid DNA has been shown to lead to geneexpression, the overall level of expression is much lower than witheither viral or liposomal vectors. Naked DNA is also generally thoughtto be unsuitable for systemic administration due to the presence ofserum nucleases. As a result, direct injection of plasmid DNA appears tobe limited to only a few applications involving tissues that are easilyaccessible to direct injection such as skin and muscle cells.

SUMMARY OF THE INVENTION

The present invention provides compositions, systems, kits, and methodsfor expression of a protein or proteins and/or biologically activenucleic acid molecule(s) in a subject (e.g., at therapeutic levels forthe extended periods of time required to produce therapeutic effects inthe host). In certain embodiments, systems and kits are provided thatcomprise a first composition comprising a first amount of polycationicstructures (e.g., empty cationic liposomes, empty cationic micelles, orempty cationic emulsions), and a second composition comprising atherapeutically effective amount of expression vector(s) (e.g.,non-viral expression vectors not associated with liposomes) that areCpG-free or CpG-reduced, where the expression vectors comprise a firstnucleic acid sequence encoding: i) a first therapeutic protein (ornon-therapeutic protein, such as a marker protein), and/or ii) a firstbiologically active nucleic acid molecule. In certain embodiments, theexpression vector comprises a second, third, or fourth nucleic acidsequence encoding a second, third, and/or fourth therapeutic ornon-therapeutic protein, and/or a second, third, or fourth biologicallyactive nucleic acid molecule. In some embodiments, the first nucleicacid sequences further encode a second, third, fourth, fifth, and/orsixth therapeutic protein, and/or a second, third, fourth, fifth, and/orsixth biologically active nucleic acid molecule. In other embodiments,such first and second compositions are sequentially administered (e.g.,systemically) to a subject such that the therapeutic protein(s) and/orthe biologically active nucleic acid molecule(s) is/are expressed in thesubject (e.g., at a therapeutic level, for at least 5 or at least 50days, or at least 100 . . . 200 . . . or at least 400 days, such thatdisease(s) or condition(s) is/are treated or physiological trait(s)is/are altered).

In some embodiments, provided herein are methods of expressing a firsttherapeutic protein and/or a biologically active nucleic acid moleculein a subject (e.g., human or non-human mammal) comprising: a)administering (e.g., systemically) a first composition to a subject,wherein the first composition comprises first amount of polycationicstructures (e.g., empty cationic liposomes, empty cationic micelles, orempty cationic emulsions) and wherein the first composition is free, oressentially free, of nucleic acid molecules (e.g., nucleic acid isundetectable or barely detectable in the composition); and b)administering (e.g., systemically, intravascularly, etc.) a secondcomposition to the subject (e.g., initiating within about 2 . . . 10 . .. 50 . . . 100 . . . 200 . . . 300 . . . 400 minutes of administeringthe first composition), wherein the second composition comprises anamount of expression vectors (e.g., non-viral expression vectors notassociated with liposomes or any other carrier), wherein the expressionvectors are CpG-free or CpG-reduced, wherein each of the expressionvectors comprise nucleic acid sequence(s) encoding: i) first, second,third, fourth, fifth, and/or sixth therapeutic protein(s) ornon-therapeutic, and/or ii) first, second, third, fourth, fifth, and/orsixth biologically active nucleic acid molecule(s). In certainembodiments, as a result of the administering the first composition andthe administering the second composition, the first therapeutic ornon-therapeutic protein and/or the biologically active nucleic acidmolecule is/are expressed in the subject (e.g., at a therapeutic level,for at least 5 . . . 50 . . . 100 . . . 300 days . . . 400 days orlonger, with respect to a disease or condition, or at an effective levelsufficient to alter a physiological or disease trait). In certainembodiments, the polycationic structures (e.g., empty liposomes) presentin the first composition have a z-average diameter of about 20-85 nm(e.g., 20 . . . 25 . . . 30 . . . 40 . . . 45 . . . 50 . . . 55 . . . 60. . . 65 . . . 70 . . . 75 . . . 80 . . . 85 nm). In certainembodiments, the polycationic structures are empty liposomes with az-average diameter of about 72-76 nm, and are small uni-lammellarvesicles.

In some embodiments, provided herein are methods of expressing a firsttherapeutic or non-therapeutic protein and/or a biologically activenucleic acid molecule in a subject comprising: a) administering a firstcomposition to a subject, wherein the subject has at least one symptomof a disease or condition, or has at least physiological trait to bealtered, wherein the first composition comprises a first amount ofpolycationic structures (e.g., empty cationic liposomes, empty cationicmicelles, or empty cationic emulsions), and wherein the firstcomposition is free, or essentially free, of nucleic acid molecules; andb) administering (or initiating administration of) a second compositionto the subject within about 100 minutes or about 200 . . . or 400minutes of administering said first composition, wherein the secondcomposition comprises a therapeutically effective amount of expressionvectors, wherein the expression vectors are CpG-free or CpG-reduced,wherein the expression vectors each comprise a first nucleic acidsequence encoding: i) a first therapeutic or non-therapeutic protein,and/or ii) a first biologically active nucleic acid molecule, c)administering dexamethasone palmitate and/or neutral lipids to thesubject, either in said first and/or second composition, or present in athird composition (e.g., within 100 or 200 . . . or 400 minutes ofadministration of the first or second compositions). In someembodiments, as a result of the administering the first composition, theadministering the second composition, and the administering of thedexamethasone palmitate and/or neutral lipids, the first therapeuticprotein and/or the biologically active nucleic acid molecule is/areexpressed in the subject at a therapeutic level with respect to thedisease or condition, or at an effective level sufficient to alter thephysiological or disease trait.

In certain embodiments, dexamethasone palmitate is in the firstcomposition, and wherein 2.0% to 6.0% (e.g., 2.0% . . . 2.5% . . . 3.0%)of the first composition comprises the dexamethasone palmitate. Incertain embodiments, the dexamethasone palmitate is administered in thethird composition, which is administered before the first and/or secondcomposition is administered, or is administered after the first and/orsecond composition, but within 100 . . . 400 minutes thereof. In certainembodiments, the methods further comprise d) administering dexamethasoneto the subject, either in the first and/or second and/or thirdcomposition, or present in a fourth composition (e.g., initiating within100 or 300 minutes of administration of the first or second or thirdcompositions, such as before any of the administrations or after theother administrations). In certain embodiments, the polycationicstructures (e.g., empty liposomes) present in the first composition havea z-average diameter of about 20-85 nm (e.g., 20 . . . 25 . . . 30 . . .40 . . . 45 . . . 50 . . . 55 . . . 60 . . . 65 . . . 70 . . . 75 . . .80 . . . 85 nm). In certain embodiments, the polycationic structures areempty liposomes with a z-average diameter of about 72-76 nm, and aresmall uni-lammellar vesicles. In some embodiments, A) the ratio is 10:1to 18:1; B) 2.0% to 6.0% of the first composition comprisesdexamethasone or dexamethasone palmitate; and/or C) each of theexpression vectors each comprise only a single expression cassette(i.e., no other expression cassettes are present in each vector),wherein the expression cassette comprises the first nucleic acidsequence encoding the first therapeutic protein and a second nucleicacid sequence encoding a second therapeutic protein, and wherein theexpression cassette encodes a self-cleaving peptide sequence (or othercleavage sequence) between the first and second nucleic acid sequences.In certain embodiments, the self-cleaving peptide comprises FMDV 2A. Inparticular embodiments, the first therapeutic protein comprises amonoclonal antibody light chain and the second therapeutic proteincomprises a heavy chain of said monoclonal antibody (e.g., the light andheavy chains combine to form an monoclonal antibody fragment (e.g., Fab)or monoclonal antibody when expresses in a subject). In certainembodiments, the polycationic structures comprise empty liposomes. Inparticular embodiments, the empty liposomes present in said firstcomposition have an average diameter of about 50-85 nm. In certainembodiments, the methods further comprise administering an agent oradditional regulating expression vectors, either in said first and/orsecond composition, or present in a third composition, wherein the agentincreases or decreases the expression at the therapeutic or effectivelevel, and/or the length of time of the expression at said therapeuticor effective level, compared to when the drug agent is not administeredto said subject (e.g., for therapeutics that need to be expressed foronly a certain, limited amount of time). In particular embodiments, theagent is selected from colchicine, dexamethasone, dexamethasonepalmitate, neutral lipids, valproic acid, theophylline, sildenafil,amlexanox, chloroquine, SAHA, and L-arginine+sildenafil.

In some embodiments, the expression vectors each further comprise aregulating nucleic acid sequence, wherein the regulating nucleic acidsequence reduces the duration of expression of the first nucleic acidsequence that would occur in the absence of said regulating nucleic acidsequence. In other embodiments, the regulating nucleic acid sequence isselected from the group consisting of: a promoter, an enhancer, a secondnucleic acid sequence encoding a second protein, and/or a secondbiologically active nucleic acid molecule. In additional embodiments,the first amount of polycationic structures in the first compositioncomprises a mixture of at least a first and second different types ofcationic liposomes that reduces the expression of the first therapeuticprotein and/or first biologically active nucleic acid molecule comparedto such expression when only said first or only said second type ofcationic liposomes are employed in said method. In particularembodiments, the therapeutic protein is expressed at a level that isabove 1 ug/ml (e.g., 1.1-1.5 ug/ml), and wherein said therapeuticprotein is expressed at the level in said subject for at least 7consecutive days (e.g., at least 7 . . . 21 . . . 50 . . . 100 . . . or400 days).

In certain embodiments, provided herein are methods of expressing afirst therapeutic protein and/or a biologically active nucleic acidmolecule in a subject comprising: a) administering (e.g., systemically)a first composition to a subject, wherein the subject has at least onesymptom of a disease or condition, or has at least physiological traitto be altered (e.g., level of hematopoietic stem cells), wherein thefirst composition comprises a first amount of polycationic structures(e.g., empty cationic liposomes, empty cationic micelles, or emptycationic emulsions), and wherein the first composition is free, oressentially free, of nucleic acid molecules; and b) administering (e.g.,systemically) a second composition to the subject be initiated (orcompleted) within about 2 . . . 10 . . . 25 . . . 100 . . . 200 or 400minutes of administering the first composition, wherein the secondcomposition comprises a therapeutically effective amount of expressionvectors (e.g., plasmid), wherein the expression vectors are CpG-free orCpG-reduced (e.g., the nucleic acid sequence of the expression vectorhas been altered to contain fewer CpG di-nucleotides than normallypresent in the wild-type version of the sequences in the vector),wherein the expression vectors each comprise nucleic acid sequence(s)encoding: i) a first therapeutic protein (or first and secondtherapeutic proteins, or first, second, and third therapeutic proteins,etc.), and/or ii) a first biologically active nucleic acid molecule (orfirst and second or more biologically active nucleic acid molecules),and wherein, as a result of the administering the first composition andthe administering the second composition, and wherein, as a result ofadministering the first and second compositions, the therapeuticprotein(s) and/or the biologically active nucleic acid molecule(s)is/are expressed in the subject at a therapeutic level with respect tothe disease or condition, or at an effective level sufficient to alterthe physiological or disease trait.

In certain embodiments, the expression vectors are not associated withpolycationic structures (e.g., empty cationic liposomes, empty cationicmicelles, or empty cationic emulsions)), or other molecules, in thesecond composition (and there are no detectable polycationic structurespresent in the second composition). In other embodiments, the expressionvectors are naked, non-viral, expression vectors (e.g., plasmids). Incertain embodiments, the expression vectors are viral expression vectors(e.g., adeno-associated viral vector or adenovirus vector or syntheticmRNA, miRNA, ribozyme or shRNA nucleic acid vectors). In particularembodiments, the first and/or second composition is administeredsystemically, regionally, transcutaneously, intradermally, orally,intramuscularly, intravenously, into the gastrointestinal tract, bladderor by pulmonary inhalation, or by an intrathecal or intraventricularroute.

In certain embodiments, the therapeutic protein or proteins and/orbiologically active nucleic acid molecule or molecules is/are expressedat the therapeutic or effective level in the subject on consecutive daysfor at least 5 . . . 20 . . . 63 . . . 100 . . . 200 . . . 300 days . .. 1 year or more. In some embodiments, the methods further comprise: c)testing the subject (e.g., body imaging or scanning), or a sample (e.g.,blood, serum, plasma, tissue, urine, etc.) from the subject, after atleast 5 . . . 20 . . . 63 . . . 100 . . . 200 . . . 300 days . . . or 1year from the administering the first and second compositions, anddetermining that the therapeutic protein(s) and/or biologically activenucleic acid molecule(s) is/are being expressed in the subject at thetherapeutic or effective level (e.g., therapeutic levels have beensustained in the subject for a time period required to producetherapeutic effects in the subject due the single treatment of the firstand second compositions). In additional embodiments, the methods furthercomprise: d) generating a written and/or electronic report thatindicates the therapeutic protein and/or biologically active nucleicacid molecule is/are being expressed in the subject at the therapeuticor effective level (e.g., for a certain amount of time). In otherembodiments, the report is sent to the treating clinician orpractitioner and/or patient from a lab that conducted the test.

In some embodiments, the therapeutic protein and/or biologically activenucleic acid molecule is/are expressed at a level of at least 50 pg/ml .. . 100 . . . 500 . . . 1000 . . . 1500 . . . 4000 . . . 8000 . . . 9500. . . 1,000,000 pg/ml (1 ug/ml) . . . 1.5 ug/ml or higher, and wherein ablood, serum, or plasma sample (or other biological sample) from thesubject is assayed to determine that the therapeutic or effective levelis achieved for at least 5 . . . 7 . . . 10 . . . 25 . . . 45 . . . 63 .. . 150 . . . 300 days, or longer, after the administration of the firstand second compositions. In other embodiments, the therapeuticprotein(s) is/are expressed at a level that is at least 50 pg/ml or atleast 100 pg/ml or at least 500, 1,000,000 pg/ml (1 ug/ml) . . . 1.5ug/ml or higher, and wherein the therapeutic protein is expressed at thelevel in the subject for at least 5 . . . 7 . . . 10 . . . 25 . . . 45 .. . 63 . . . 150 . . . 300 . . . 350 consecutive days. In certainembodiments, the therapeutic protein and/or biologically active nucleicacid molecule is expressed (e.g., at therapeutic levels) in the subjectwithout clinically significant elevated toxicity (e.g., as measured byALT (alanine aminotransferase) and/or AST (aspartate aminotransferase))after at least 48 hours following the administration of the first andsecond compositions.

In certain embodiments, the therapeutic protein is human G-CSF (e.g., asencoded by SEQ ID NO:1, or sequence with at least 98% identity with SEQID NO:1) and is expressed in the subject at a therapeutic level of atleast 100 pg/ml as measured in a blood, serum, or plasma sample, whereinthe therapeutic protein is expressed in the subject for at least sevendays, and wherein the disease, condition, or physiological trait isselected from the group consisting of: neutropenia caused bychemotherapy, non-elevated levels of hematopoietic stem cells in bloodof a stem cell donor or recipient, heart degeneration, cerebralischemia, amyotrophic lateral sclerosis, neutrophil deficiency diseases,and radiation exposure. In particular embodiments, the G-CSF isexpressed for at least 5, or 6, or 7 days, but no more than about 10days (e.g., using drugs, promoter/enhancer combinations, additionalexpression cassette within the nucleic acid vector or additionalexpressed proteins to limit production to about 10 days to avoid anytoxic neutrophilia-related side effects by expression beyond about 10days). In other embodiments, the therapeutic protein is Rituximab orsimilar anti-CD20 antibody or antibody fragment. In some embodiments,the therapeutic protein is human Factor IX or similar protein.

In particular embodiments, the therapeutic protein or proteins and/orbiologically active nucleic acid molecule or molecules is/are expressedin the subject for a sufficient amount of time at the therapeutic levelto reduce or eliminate the at least one symptom (or all symptoms)without the subject having to receive any other treatment that providesthe therapeutic protein(s) and/or biologically active nucleic acidmolecule(s) to the subject. In further embodiments, during thesufficient time, the subject does not receive any other specifictreatment (e.g., no other specific therapeutic treatment that providesthe therapeutic protein or biologically active nucleic acid molecule(s)to the subject). In certain embodiments, the subject has multiplesymptoms of a disease or diseases, and wherein the sufficient amount oftime is such that all or substantially all of the multiple symptoms ofthe disease(s) and/or the condition(s) are reduced or eliminated in thesubject (e.g., permanently, or for at least 20 days . . . 50 days . . .200 days . . . 1 year or longer). In other embodiments, during thesufficient time, the subject does not receive the any otherdisease-specific treatment.

In some embodiments, the first amount of the polycationic structures(e.g., empty cationic liposomes, empty cationic micelles, or emptycationic emulsions) is about 0.01-70, 30-50, or 20-60, μmoles per 1kilogram of the subject (e.g., 0.01 . . . 1 . . . 10 . . . 20 . . . 40 .. . or 60 μmoles per kilogram). In other embodiments, the ratio of thefirst amount of the polycationic structures (e.g., empty cationiclipids) to the therapeutically effective amount of the expressionvectors is 0.5:1 to 25:1, nmoles of polycationic structures (e.g., emptycationic lipids) to 1 μg of expression vectors (e.g., 0.5:1 . . . 1:1 .. . 4:1 . . . 8:1 . . . 12:1 . . . 17:1 . . . 21:1 . . . or 25:1). Incertain embodiments, the ratio of the first amount of the polycationicstructures (e.g., empty cationic lipids) to the therapeuticallyeffective amount of the expression vectors is 7:1 to 13:1, nmoles ofpolycationic structures (e.g., empty cationic lipids) to 1 μg ofexpression vectors. In particular embodiments, the therapeuticallyeffective amount of the expression vectors is 0.001-8.0 milligrams ofthe expression vectors per 1 kilogram of the subject (e.g., 0.001 . . .0.1 . . . 3.0 . . . 4.5 . . . 5.7 . . . 7.1 . . . 8.0 milligrams perkilogram). In some embodiments, the therapeutically effective amount ofexpression vectors is 0.001 to 1 μg per 1 kilogram of the subject (e.g.,0.001 . . . 0.01 . . . 0.1 . . . 1 μg per kilogram of subject). Incertain embodiments, the therapeutically effective amount of theexpression vectors is about 0.01-4.0 milligrams of the expressionvectors per 1 kilogram of the subject.

In some embodiments, the first nucleic acid sequence encodes the firstor first and second, or first, second, and third, therapeuticprotein(s). In additional embodiments, the first nucleic acid sequenceencodes the biologically active nucleic acid molecule(s). In otherembodiments, the subject is a human. In additional embodiments, theexpression vectors are CpG-free. In other embodiments, the expressionvectors are CpG-reduced. In other embodiments, the therapeuticprotein(s) is/are human protein(s) or animal protein(s).

In some embodiments, the polycationic structures do not containcholesterol (e.g., cholesterol free empty cationic micelles orliposomes). In certain embodiments, the cationic liposomes each compriseat least 60% DOTAP and/or DPTAP (e.g., 60% . . . 75% . . . 85% . . . 95%. . . 98% . . . 100% DOTAP and/or DPTAP). In other embodiments, all orsubstantially all of the cationic liposomes are multi-lamellar vesicles.In further embodiments, all or substantially all of the cationicliposomes are uni-lamellar vesicles. In further embodiments, thecationic liposomes each comprise at least 99% DOTAP or 99% DPTAP. Infurther embodiments, the empty cationic liposomes each comprise DOTAPand cholesterol. In additional embodiments, the cationic liposomes eachcomprise about one-third cholesterol and about two-thirds DOTAP and/orDPTAP. In further embodiments, the first nucleic acid sequence encodeshuman G-CSF (e.g., as shown in SEQ ID NO:1).

In certain embodiments, the biologically active nucleic acid molecule(s)comprises sequence(s) selected from: shRNA sequence(s), miRNAsequence(s), antisense sequence(s), ribozyme(s), and/or CRISPR singleguide RNA sequence(s) (sgRNA). In other embodiments, the CRISPR sgRNAcomprises: i) a Cas9 nuclease-recruiting sequence (tracRNA), and ii) atarget-specific sequence (crRNA) that hybridizes to a sgRNA target site.In particular embodiments, the biologically active nucleic acid moleculetargets human p65 (aka, NF-kappa-B p65 or RELA).

In further embodiments, each of the expression vectors further comprisesa second nucleic acid sequence encoding: i) a second therapeuticprotein, and/or ii) a second biologically active nucleic acid molecule.In some embodiments, each of the expression vectors further comprises athird nucleic acid sequence encoding: i) a third, and/or fourththerapeutic protein, and/or ii) a third, and/or fourth biologicallyactive nucleic acid molecule. In further embodiments, each of theexpression vectors further comprise a first promoter associated with thefirst nucleic acid sequence, and a second promoter associated with thesecond nucleic acid sequence, and wherein the first and second promotersare the same or different. In other embodiments, the therapeutic oreffective expression level of the first nucleic acid sequence and/or thelength of time of the therapeutic or effective expression level, isreduced compared to the expression level or the length of time, when thesecond nucleic acid is not present and/or expressed from the expressionvectors. In other embodiments, the first nucleic acid sequence isexpressed at the therapeutic level for at least 5 days, but less than 21days (e.g., 5 . . . 7 . . . 13 . . . 16 . . . 20 . . . and 21 days). Incertain embodiments, the first nucleic acid sequence encodes thetherapeutic protein, and wherein the therapeutic protein comprises humanG-CSF.

In other embodiments, the expression vector provides the expression atthe therapeutic or effective level for a first length of time and/or ata first level of expression when each of the expression vectorscomprises a first promoter and first enhancer associated with the firstnucleic acid sequence, and wherein the first length of time and/orexpression level is altered when a second promoter, different from thefirst promoter, replaces the first promoter, and/or a second enhancer,different from the second enhancer, replaces the second promoter, on theexpression vectors. In other embodiments, the expression at thetherapeutic or effective level for a first length of time is for atleast 10 . . . 15 . . . 45 . . . 100 . . . 200 . . . 300 days, andwherein replacement with the second promoter and/or second enhancerreduces expression at the therapeutic or effective level to a secondlength of time that is less than 10 . . . 15 . . . 45 . . . 100 . . .200 days. In other embodiments, each of the expression vectors comprisesa first promoter and a first enhancer, and wherein the first promoterand the first enhancer cause expression at the therapeutic level for atleast 5 days, but less than 21 . . . 15 . . . or 10 days. In particularembodiments, the first nucleic acid sequence encodes the therapeuticprotein, and wherein the therapeutic protein comprises human G-CSF.

In some embodiments, the methods further comprise administering a drugagent or agents, either in the first and/or second composition, orpresent in a third composition, wherein the drug agent or agentsincrease or decrease the expression of the first nucleic acid (e.g., atthe therapeutic or effective level, and/or the length of time of theexpression at the therapeutic or effective level), compared to when thedrug agent or agents are not administered to the subject. In particularembodiments, the drug agent increases the expression level of the firstnucleic acid in the subject, and wherein the drug is selected fromcolchicine, an immunosuppressant, dexamethasone, dexamethasonepalmitate, sildenafil, or L-arginine+sildenafil. In certain embodiments,the drug (e.g., dexamethasone or dexamethasone palmitate) is present atbetween 2.0% and 6.0% of a polycationic structure (e.g., empty cationiclipid composition), such as at 2.0% . . . 2.5% . . . 3.5% . . . 4.5% or6.0%. In other embodiments, the drug (e.g., dexamethasone ordexamethasone palmitate), is administered to the subject before or afterthe polycationic structure and vector compositions are administered. Incertain embodiments, the polycationic structures (e.g., empty liposomes)present in the first composition have a z-average diameter of about20-85 nm (e.g., 20 . . . 25 . . . 30 . . . 40 . . . 45 . . . 50 . . . 55. . . 60 . . . 65 . . . 70 . . . 75 . . . 80 . . . 85 nm). In certainembodiments, the polycationic structures are empty liposomes with az-average diameter of about 72-76 nm, and are small uni-lammellarvesicles.

In other embodiments, the therapeutic protein is expressed at a level ofat least two times higher (or at least 3 or 4 or 5 times higher) whenthe drug agent is administered to the subject compared to when the drugagent is not administered to the subject. In particular embodiments, thedrug agent decreases the expression level of the first nucleic acidsequence, and wherein the drug agent is L-arginine. In furtherembodiments, the therapeutic protein is expressed at a level of at leasttwo times (or at least three times or four times) lower when the drugagent is administered to the subject compared to when the drug agent isnot administered to the subject. In some embodiments, the drug agentcomprises an anti-inflammatory agent. In additional embodiments, thedrug agent is selected from the group consisting of: amlexanox,chloroquine, valproic acid, theophylline, DHA, prostaglandin, and SAHA.

In further embodiments, the expression vectors are free of operablematrix attachment region (MAR) sequences. In certain embodiments, theexpression vectors are free of operable EBNA-1 and/or EBV viralsequences. In certain embodiments the subject's blood pressure,immediately prior to said administering said first and secondcompositions, is not altered (e.g., no physical transfection aids areapplied to the subject to attempt to increase expression of the firstnucleic acid sequence).

In particular embodiments, the therapeutic level and/or effective levelis at least 150 . . . 100 . . . 500 . . . 1000 . . . 1500 . . . 5000 . .. 1,000,000 pg/ml (1 ug/ml) . . . 1.5 ug/ml or higher, and wherein ablood, serum, or plasma sample (or other biological sample) from thesubject is determined to be at the therapeutic level and/or effectivelevel at least 7 . . . 10 . . . 25 . . . 45 . . . 63 . . . 150 . . . 300. . . 400 days or more after the administration of the first and secondcompositions. In particular embodiments, the sample from the subject istested with an ELISA assay or by mass spectrometry to determine theexpression level.

In some embodiments, the methods further comprise administering atherapeutically effective amount of neutral liposomes to the subject,wherein the neutral liposomes are present in the first and/or secondcomposition, and/are administered in a third composition, and whereinthe therapeutically effective amount of neutral liposomes areadministered to the subject prior to the administering the secondcomposition. In certain embodiments, the neutral liposomes comprise atleast material selected from: phospholipon 90H, hydrogenated soy PC,stearic and palmitic. In other embodiments, the therapeuticallyeffective amount of neutral liposomes are present in the firstcomposition or present in a third composition administered to thesubject. In further embodiments, the neutral liposomes are multilamellarvesicles or extruded to 0.2 or 0.1 um. In particular embodiments,administering the therapeutically effective amount of the neutralliposomes causes expression of the first therapeutic protein and/or thebiologically active nucleic acid molecule in the subject that is atleast 3 . . . 4 . . . 25 . . . 100 . . . 350 . . . or 600 times higherthan occurs when the neutral liposomes are not administered to thesubject. In certain embodiments, the ratio of empty cationic liposomesto the neutral liposomes administered to the subject is between about2:1 and 1:5 (e.g., 2:1 . . . 1:1 . . . 2:5 . . . 1:5).

In some embodiments, provided herein are methods of expressing a firsttherapeutic protein and/or a biologically active nucleic acid moleculein a subject comprising: a) administering a first composition to asubject, wherein the first composition comprises an anti-inflammatoryagent; and b) administering or initiating administration of, a secondcomposition to the subject within about 2 minutes . . . 20 minutes . . .1 hour . . . 24 hours . . . 5 days . . . 7 days . . . 9 days or more ofadministering the first composition, wherein the second compositioncomprises a therapeutically effective amount of polyplexes, wherein eachpolyplex comprises an expression vector and polyethylenimine, whereinthe expression vector is CpG-free or CpG-reduced, wherein eachexpression vector comprises a first nucleic acid sequence encoding: i) afirst therapeutic protein (and/or first and second proteins), and/or ii)a first (and/or first and second) biologically active nucleic acidmolecule, and wherein, as a result of administering the firstcomposition and administering the second composition, the firsttherapeutic protein and/or the biologically active nucleic acid moleculeis/are expressed in the subject. In further embodiments, the subject hasat least one symptom of a disease or condition, or has at least onephysiological trait desired to be altered, and wherein the firsttherapeutic protein and/or the biologically active nucleic acid moleculeis expressed at a therapeutic level with respect to the disease,condition, or physiological trait to be altered. In some embodiments,the anti-inflammatory agent is selected from the group consisting ofamlexanox, chloroquine, and suberanilohydroxamic acid (SAHA).

In some embodiments, the expression vector comprises a plasmid or othernon-viral vector. In further certain embodiments, the administration instep b) is accomplished by systemically administering the secondcomposition.

In some embodiments, provided herein are systems or kits comprising: a)a first composition comprising a first amount of polycationic structures(e.g., empty cationic liposomes, empty cationic micelles, or emptycationic emulsions), wherein the first composition is free, oressentially free, of nucleic acid molecules; and b) a second compositioncomprises a therapeutically effective amount of expression vectors(e.g., non-viral and not associated with liposomes or other carriermolecules), wherein the expression vectors are CpG-free or CpG-reduced,wherein each of the expression vectors comprises a first nucleic acidsequence encoding: i) a first therapeutic protein or non-therapeuticprotein, and/or ii) a first biologically active nucleic acid molecule.In other embodiments, the expression vectors are a naked, non-viralexpression vectors (e.g., plasmid). In certain embodiments, at least oneof the following applies: i) wherein the ratio of the first amount ofthe polycationic structure (e.g., empty cationic liposome) to thetherapeutically effective amount of expression vectors is 2:1 to 25:1 or5:1 to 25:1; ii) wherein 2.0% to 6.0% of the first composition comprisesdexamethasone palmitate; iii) wherein the first composition furthercomprises neutral lipid, and iv) wherein the polycationic structurescomprise empty liposomes, and wherein the empty liposomes present in thefirst composition have a z-average diameter of about 20-85 nm (e.g., 20. . . 25 . . . 30 . . . 40 . . . 45 . . . 50 . . . 55 . . . 60 . . . 65. . . 70 . . . 75 . . . 80 . . . 85 nm). In certain embodiments, thevectors are viral vectors (e.g., AAV or adeno viral vectors). Inparticular embodiments, the therapeutic protein is human G-CSF (e.g., asshown in SEQ ID NO:1).

In particular embodiments, the first amount of the polycationicstructure (e.g., empty cationic liposomes) is between 0.1 to 7.0millimoles (e.g., 0.1 . . . 5.0 . . . 7.0 millimoles) or 1.5 and 5.0millimoles (e.g., suitable amount for administration to a humansubject). In other embodiments, the ratio of the first amount of thepolycationic structure (e.g., empty cationic liposome) to thetherapeutically effective amount of the expression vectors is 0.5:1 to25:1, nmoles of empty cationic lipid to 1 μg of expression vectors(e.g., 0.5:1 . . . 1:1 . . . 5:1 . . . 10:1 . . . 15:1 . . . 25:1). Insome embodiments, the ratio of the first amount of the polycationicstructure (e.g., empty cationic lipid) to the therapeutically effectiveamount of the expression vectors is 7:1 to 13:1, nmoles of polycationicstructure to 1 μg of expression vectors (e.g., 7:1 . . . 10:1 . . . or13:1). In other embodiments, the therapeutically effective amount of theexpression vectors is between 0.1 and 800 milligrams (e.g., suitableamount for administration to a human subject, such as when the vector isa plasmid). In certain embodiments, the amount is 1 . . . 25 . . . 400 .. . or 800 milligrams of expression vectors for human administration.

In other embodiments, the first nucleic acid sequence encodes the firsttherapeutic protein. In additional embodiments, the first nucleic acidsequence encodes the biologically active nucleic acid molecule. Inparticular embodiments, the expression vectors are CpG-free. In otherembodiments, the expression vectors are CpG-reduced. In furtherembodiments, the first therapeutic protein is a human protein. In otherembodiments, the first nucleic acid sequence encodes the therapeuticprotein, and wherein the therapeutic protein comprises human G-CSF,Rituximab, a monoclonal antibody or monoclonal antibody fragment (e.g.,Fab), or human Factor IX.

In certain embodiments, the empty cationic liposomes, micelles, oremulsions, each comprise at least 60% DOTAP and/or DPTAP (e.g., 60% . .. 75% . . . 85% . . . 95% . . . 98% . . . 100% DOTAP and/or DPTAP), andmay be cholesterol-free (e.g., no detectable cholesterol in thecomposition). In other embodiments, all or substantially all of theempty cationic liposomes, micelles, or emulsions are multilamellarvesicles. In further embodiments, all or substantially all of the emptycationic liposomes, micelles, or emulsions are either unilamellar,multilamellar, or oligolamellar vesicles. In further embodiments, theempty cationic liposomes, micelle, or emulsions each comprise at least99% DOTAP or at least 99% DPTAP, and may be cholesterol free. In furtherembodiments, the empty cationic liposomes each comprise DOTAP and/orDPTAP and cholesterol. In additional embodiments, the empty cationicliposomes, micelles, or emulsions each comprise about one-thirdcholesterol and about two-thirds DOTAP and/or DPTAP.

In certain embodiments, the first biologically active nucleic acidmolecule comprises a sequence selected from: an siRNA or shRNA sequence,a miRNA sequence, an antisense sequence, and a CRISPR single guide RNAsequence (sgRNA). In other embodiments, the CRISPR sgRNA comprises: i) aCas9 nuclease-recruiting sequence (tracRNA), and ii) a target-specificsequence (crRNA) that hybridizes to a sgRNA target site.

In further embodiments, each of the expression vectors further comprisesa second nucleic acid sequence encoding: i) a second therapeuticprotein, and/or ii) a second biologically active nucleic acid molecule.In further embodiments, each of the expression vectors further comprisea first promoter associated with the first nucleic acid sequence, and asecond promoter associated with the second nucleic acid sequence, andwherein the first and second promoters are the same or different.

In some embodiments, the kits and systems further comprise a firstcontainer and a second container, and wherein the first composition ispresent in the first container and the second composition is present inthe second container. In other embodiments, kits and systems furthercomprise a packaging component (e.g., cardboard box, plastic pouch,etc.), wherein the first container and the second container are insidethe packaging component.

In certain embodiments, the kits and systems further comprise a drugagent or drug agents, wherein the drug agent(s) are present in the firstand/or second compositions, or is present in a third composition. Inadditional embodiments, the drug agent is selected from colchicine, animmunosuppressant, dexamethasone, sildenafil, L-arginine, orL-arginine+sildenafil. In further embodiments, the drug agent comprisesan anti-inflammatory agent. In further embodiments, the drug agent isselected from the group consisting of: amlexanox, valproic acid,theophylline, chloroquine, and SAHA.

In particular embodiments, the expression vectors are free of operablematrix attachment region (MAR) sequences. In additional embodiments, theexpression vectors are free of operable EBNA-1 and/or EBV viralsequences.

In certain embodiments, the kits and systems further comprise atherapeutically effective amount of neutral liposomes, wherein theneutral liposomes are present in the first and/or second compositions,or is present in a third composition. In additional embodiments, thetherapeutically effective amount of neutral liposomes are present in thefirst composition. In other embodiments, the neutral liposomes aremultilamellar or oligo- or uni-lamellar vesicles. In furtherembodiments, the ratio of empty cationic liposomes or micelles to theneutral liposomes is between about 2:1 and 1:5 (e.g., 2:1 . . . 1:1 . .. 3:5 . . . 1:5).

In some embodiments, provided herein are a first composition and asecond, separate, composition for combined use in the treatment of adisease amenable to treatment with in vivo expression of a firsttherapeutic protein and/or biologically active nucleic acid molecule,wherein the first composition comprises a first amount of polycationicstructures (e.g., empty cationic liposomes, empty cationic micelles, orempty cationic emulsions), wherein the first composition is free, oressentially free, of nucleic acid molecules; and b) a second compositionthat comprises a therapeutically effective amount of expression vectors,wherein the expression vectors are CpG-free or CpG-reduced, wherein eachof the expression vectors comprises a first nucleic acid sequenceencoding: i) a first therapeutic protein, and/or ii) a firstbiologically active nucleic acid molecule.

In certain embodiments, provided herein are methods of expressing afirst therapeutic protein and/or a biologically active nucleic acidmolecule in a subject comprising: a) administering a first compositionto a subject, wherein the first composition comprises a first amount ofpolycationic structures (e.g., empty cationic liposomes, empty cationicmicelles, or empty cationic emulsions), and wherein the firstcomposition is free, or essentially free, of nucleic acid molecules; andb) administering a second composition to the subject within about 100minutes or 200 minutes of administering the first composition, whereinthe second composition comprises a therapeutically effective amount ofnon-viral expression vectors, wherein the expression vectors areCpG-free or CpG-reduced, wherein the expression vectors each comprise afirst nucleic acid sequence encoding: i) a first therapeutic protein,and/or ii) a first biologically active nucleic acid molecule, andwherein, as a result of administering the first composition andadministering about the second composition, the first therapeuticprotein and/or the biologically active nucleic acid molecule is/areexpressed in the subject at a level above (e.g., at least 150 . . . 300. . . 575 . . . 1000 . . . 1500 . . . 2000 . . . 5000 . . . or 1,000,000pg/ml) (e.g., as measured in a serum sample from the subject (e.g.,after 7 . . . 25 . . . 50 days from the first and secondadministrations).

In certain embodiments, provided herein are methods comprising:administering a composition to a subject comprising a therapeuticallyeffective amount of non-viral expression vectors that are CpG-free orCpG-reduced and comprise a first nucleic acid sequence encoding: i) afirst therapeutic protein, and/or ii) a first biologically activenucleic acid molecule, and wherein, as a result of administering thefirst and second compositions, the first therapeutic protein and/or thebiologically active nucleic acid molecule is/are expressed in thesubject at a level above 100 pg/ml (e.g., at least 150 . . . 400 . . .1200 . . . 2000 . . . 5000 . . . or more than 1,000,000 pg/ml) (e.g., asmeasured in a serum sample from the subject (e.g., after 7 . . . 25 . .. 50 days from the first and second administrations).

In certain embodiments, the polycationic structures comprises emptycationic liposomes, micelles, or emulsions. In other embodiments, thepolycationic structures comprises one or more of the following, eitheralone or combined with polycationic structures: linear or branchedpolyethyleneimine, dendrimers (e.g., 4th generation pamaam dendrimerbased on ethylene diamine, polylysine, polyarginine, and protaminesulfate), poly-lysine, and protamine sulfate. In certain embodiments,the polycationic structures are provided as a cationic emulsion. Inparticular embodiments, the surfactants in the emulsions are selectedfrom: cetylpyridinium chloride, cetyltrimethylammonium bromide or thelike. In other embodiments, the emulsions further comprise a neutralcomponent, such as tweens, spans and triglycerides. In particularembodiments, the emulsions comprises a cationic lipid, such as, forexample, DOTAP, DPTAP, DOTMA, or DDAB. In some embodiments, theemulsions are self-emulsifying emulsions or microemulsions (SEDDS,SMEDDS).

In some embodiments, provided herein are methods of expressing a firstand second proteins and/or first and second biologically active nucleicacid molecules in a subject comprising: a) administering a firstcomposition to a subject, wherein the subject has at least one symptomof a disease or condition, or has at least physiological trait to bealtered, wherein said first composition comprises a first amount ofpolycationic structures, and wherein said first composition is free, oressentially free, of nucleic acid molecules; and b) administering asecond composition to said subject within about 100 minutes ofadministering the first composition, wherein the second compositioncomprises a therapeutically effective amount of expression vectors,wherein the expression vectors are non-viral and are CpG-free orCpG-reduced, wherein the expression vectors each comprise: i) a firstexpression cassette encoding: A) a first protein, and/or B) a firstbiologically active nucleic acid molecule, and ii) a second expressioncassette encoding: A) a second protein and/or B) a second biologicallyactive nucleic acid molecule. In certain embodiments, as a result of theadministering the first composition and the administering the secondcomposition, the first and second proteins and/or said first and secondbiologically active nucleic acid molecule is/are expressed in thesubject at a therapeutic level with respect to the disease or condition,or at an effective level sufficient to alter said physiological ordisease trait.

In particular embodiments, the first protein comprises a monoclonalantibody light chain, and the second protein comprises a heavy chain ofsaid monoclonal antibody. In other embodiments, the first and secondexpression cassettes both comprise regulatory elements. In additionalembodiments, the regulatory elements are the same or different in saidfirst and second expression cassettes.

DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic representation of the various CPG-free plasmidconstructs used in Example 1.

FIG. 2 shows the CpG-free modified nucleic sequence of h-GCSF (SEQ IDNO:1) and the amino acid sequence of h-GCSF (SEQ ID NO:2). The positionswhere CpG di-nucleotides have been eliminated are shown in underline inSEQ ID NO:1.

FIG. 3 shows a graph of serum human G-CSF levels produced in mice bysequential, IV cationic liposome injection followed by IV DNA vectorinjection.

FIG. 4 shows a histogram of WBC and absolute neutrophil counts, 21 daysafter sequential, IV cationic liposome then DNA vector injection.

FIG. 5 shows serum human G-CSF levels produced in mice by sequential IVinjection of either single or dual cassette, hG-CSF single plasmidvectors.

FIG. 6 shows serum hG-CSF levels in mice, 21 days after IV injection ofcationic liposomes, then DNA containing different promoter-enhancercombinations linked to the hG-CSF gene.

FIG. 7 shows mouse lung luciferase levels, 7 days after sequential IVinjection of cationic liposomes, then single cassette, EF1-luciferaseDNA alone or together with a particular drug.

FIG. 8 shows mouse lung luciferase levels, 7 days after sequential IVinjection of cationic liposomes, then dual cassette,EF1-hG-CSF-EF1-luciferase DNA alone or with certain drug(s).

FIG. 9 shows mouse lung luciferase levels, 10 days after sequential IVinjection of cationic liposomes, then a DNA vector containing one of aseries of different promoter-enhancer combinations, each either with orwithout MARs, and all linked to the luciferase gene.

FIG. 10 shows mouse lung luciferase levels, 1 or 5 days after IVinjection of PEI:EF-1 Luc DNA complexes or sequential IV injection ofcationic liposomes, then the identical EF-1 Luc DNA.

FIG. 11 shows mouse lung luciferase levels, 1 day after sequential IVinjection of one of seven different cationic liposome formulations, thensingle cassette, EF1-luciferase DNA.

FIG. 12 shows mouse spleen luciferase levels, 1 day after IV injectionof PEI:EF-1 Luc DNA complexes alone or mixed with one of four differentdrugs.

FIG. 13 shows mouse lung luciferase levels, 1 or 7 days after sequentialIV injection of cationic liposomes, then one of a series of dualcassette, EF-1-Luc-hG-CSF DNA vectors.

FIG. 14 shows serum human G-CSF levels produced in mice, 1 day aftersequential, IV cationic liposome injection, with or without co-injectionof neutral liposomes, followed by IV injection of a dual cassette,single plasmid vector.

FIG. 15 shows serum human G-CSF levels produced in mice, 1 or 7 daysafter sequential, IV cationic liposome co-injection with neutralliposomes, followed by IV injection of a dual cassette plasmid vector.

FIG. 16 shows serum human G-CSF levels produced in mice 7 days aftersequential, IV cationic liposome injection with SUV, 0.1 μm extruded orMLV cationic liposomes, followed by IV injection of a EF1-hG-CSF plasmidvector.

FIG. 17 shows results from Example 2, wherein one sequential IVinjection of cationic liposomes followed by a CPG-free, human G-CSF DNAvector produces supra-therapeutic human G-CSF serum protein levels inmice for at least the next 428 days.

FIG. 18 shows results from Example 3, wherein it was shown that onesequential IV injection of cationic liposomes followed by a CPG-free,human G-CSF DNA vector produces supra-therapeutic human G-CSF serumprotein, WBC and ANC levels, with normal ALT (alanine aminotransferase)and AST (aspartate aminotransferase) in rats.

FIG. 19 shows results from Example 3, where it was shown that cationicliposomes generated from DPTAP mediate in vivo transfection.

FIG. 20 shows that toxicity as measured by serum levels of alanineaminotransferase (ALT) and aspartate aminotransferase (AST) are elevated2 to 5 fold at 24 hrs and return to control levels by 48 hours aftersequential injection of cationic liposomes then plasmid DNA.

FIG. 21 shows incorporation of 2.5 mole % dexamethsone palmitate (DexP)into cationic DOTAP liposomes increases expression of hG-CSF at 24 hoursafter sequential IV injection while simultaneously reducing toxicity, asmeasured by ALT levels to close to background levels.

FIG. 22 shows that IV injection of DOTAP liposomes containing 2.5%dexamethasone palmitate reduces toxicity, as measured by ALT levels tobackground levels while significantly increasing human G-CSF proteinlevels.

FIG. 23 shows pre- and post-injection of Dexamethasone significantlyincreases hG-CSF protein levels while reducing toxicity, as measured byALT levels to close to background levels.

FIG. 24 shows manipulating Lipid:DNA Ratios increases hG-CSF levelswhile reducing toxicity, as measured by ALT levels to background levels.

FIG. 25 shows that IP pre-injection of dexamethasone, followed by 2.5mole % dexamethsone palmitate in cationic DOTAP liposomes then a dualcassette, single plasmid DNA vector encoding Rituximab significantlyincreases serum Rituximab levels over time in mice.

FIG. 26 shows dexamethasone pre-injection followed by one IV sequentialinjection of DexP cationic liposomes plus neutral lipid then a dualcassette, single plasmid DNA vector encoding Rituximab produces extendedserum levels of fully functional Rituximab protein in mice.

FIG. 27 shows mouse serum tested at 6 weeks following IV sequentialinjection of a dual cassette, single plasmid Rituximab DNA vector bindstarget CD20+ human B lymphoma (Raji) cells similarly to recombinantRituximab protein.

FIG. 28 Rituximab protein in serum from Rituximab DNA vector-injectedmice induces lysis of Raji CD20+ human B cells at levels similar torecombinant Rituximab.

FIG. 29 shows IP pre-injection of dexamethasone, then neutral lipid plus2.5 mole % dexamethsone palmitate in DOTAP liposomes increases serumRituximab levels over time in rats.

FIG. 30 shows codon-optimization of Rituximab dual cassette, singleplasmid DNA vectors further increases serum Rituximab levels 24 hoursafter sequential IV injection.

FIG. 31 shows that one sequential, IV cationic liposome injection ofcodon-optimized dual cassette, single plasmid Rituximab DNA vectorsproduces extended serum Rituximab levels.

FIG. 32 shows that pre-injection of selected drugs significantlyincreases serum Rituximab levels produced by sequential IV, cationicliposome injection of a codon-optimized dual cassette, single plasmidRituximab DNA vector.

FIG. 33 shows sequential IV injection of a single cassette DNA vectorencoding the Rituximab heavy and light chains separated by a 2A selfcleaving peptide sequence produces significant serum Rituximab proteinlevels.

FIG. 34 shows that manipulating Lipid:DNA Ratios increases serumRituximab levels while reducing toxicity, as measured by ALT levels toclose to background levels.

FIG. 35 shows that pretreatment with either valproic acid ortheophylline significantly increases serum human factor nine levelsproduced by sequential IV, cationic liposome injection of acodon-optimized, EF-1-driven plasmid vector encoding a human factor IXcDNA.

FIG. 36 shows the arrangement of the Rituximab (anti-CD20) dual cassetteplasmids used in the Examples. In this figure, the followingabbreviations apply: M: Mar (M1:β-Glo, M2:21q21 and M3: IFNβ); K: KozakSequence (K1:AAGCTTTCC, SEQ ID NO:3; K2:AAGCCACC, SEQ ID NO:4);Enhancer: mCMV or hCMV; Promoter: CMV or EF1; 5′UTR: 1126 or htiv; H:Chimeric Heavy Chain cDNA; L: Chimeric Light Chain cDNA; and pA: polyA

FIG. 37 shows the arrangement of the Rituximab (anti-CD20) single(bisicontronic) plasmid used in the Examples. In this figure, thefollowing abbreviations apply: K: Kozak Sequence (K1:AAGCTTTCC K2, SEQID NO:3; AAGCCACC, SEQ ID NO:4); Enhancer: mCMV or hCMV; Promoter: CMVor EF1; 5′UTR: 1126 or htiv

H: Chimeric Heavy Chain cDNA; L: Chimeric Light Chain cDNA; F: Furin(F1: RHQR; F2: RAKR); 2A Peptide: P2A or F2A; and pA: polyA.

FIG. 38 shows the arrangement of the human Factor IX plasmids used inthe Examples. The following abbreviations apply in this figure: M: Mar(M1:β-Glo, and M3: IFNβ); Kozak2: Kozak Sequence2 (AAGCCACC, SEQ IDNO:4); Enhancer: mCMV; Promoter: EF1; 5′UTR: I126; hFIX: human Factor XIcDNA; and pA: polyA

FIG. 39 shows one example (No. 8, G4) of a bicistronic, single cassetteplasmid construct (SEQ ID NO:5) used in the Examples below thatexpresses the heavy and light chains (underlined) of Rituximab.

FIG. 40 shows one example (No. 2) of a dual cassette non-optimizedanti-CD20 CpG free plasmid construct (SEQ ID NO:6) used in the Examplesbelow that expresses the heavy and light chains (underlined) ofRituximab.

FIG. 41 shows one example (No. 4) of a dual cassette non-optimizedanti-CD20 CpG free plasmid construct (SEQ ID NO:7) used in the Examplesbelow that expresses the heavy and light chains (underlined) ofRituximab.

FIG. 42 shows one example (No. 4) of a dual cassette MAR-less optimizedanti-CD20 plasmid construct (SEQ ID NO:8) used in the Examples belowthat expresses the heavy and light chains (underlined) of Rituximab.

FIG. 43 shows one example (No. 6) of a dual cassette MAR-containingoptimized anti-CD20 plasmid construct (SEQ ID NO:9) used in the Examplesbelow that expresses the heavy and light chains (underlined) ofRituximab.

FIG. 44 shows one example (No. 4) of a plasmid construct (SEQ ID NO:10)used in the Examples below that expresses human Factor IX.

DEFINITIONS

As used herein, the phrase “CpG-reduced” refers to a nucleic acidsequence or expression vector that has less CpG di-nucleotides thanpresent in the wild-type versions of the sequence or vector. “CpG-free”means the subject nucleic acid sequence or vector does not have any CpGdi-nucleotides. An initial sequence, that contains CpG dinucleotides(e.g., wild-type version of human G-CSF), may be modified to remove CpGdinucleotides by altering the nucleic acid sequence. Such CpGdi-nucleotides can be suitably reduced or eliminated not just in acoding sequence, but also in the non-coding sequences, including, e.g.,5′ and 3′ untranslated regions (UTRs), promoter, enhancer, polyA, ITRs,introns, and any other sequences present in the nucleic acid molecule orvector.

As used herein, “empty liposomes” refers to liposomes that do notcontain nucleic acid molecules but that may contain other bioactivemolecules (e.g., liposomes that are only composed of the lipid moleculesthemselves, or only lipid molecules and a small molecule drug).

As used herein, “empty cationic micelles” refers to cationic micellesthat do not contain nucleic acid molecules but that may contain otherbioactive molecules (e.g., micelles that are only composed of lipid andsurfactant molecules themselves, or only lipid and surfactant moleculesand a small molecule drug).

As used herein, “empty cationic emulsions” refers to cationic emulsionsor microemulsions that do not contain nucleic acid molecules but thatmay contain other bioactive molecules.

DETAILED DESCRIPTION

The present invention provides compositions, systems, kits, and methodsfor generating expression of a protein or biologically active nucleicacid molecule in a subject (e.g., at therapeutic levels for extendedperiods of time). In certain embodiments, systems and kits are providedthat comprise a first composition comprising a first amount polycationicstructures (e.g., empty cationic liposomes, empty cationic micelles, orempty cationic emulsions), and a second composition comprising atherapeutically effective amount of expression vectors (e.g., non-viralexpression vectors not associated with liposomes) that are CpG-free orCpG-reduced, where the expression vectors comprise a first nucleic acidsequence encoding: i) a first therapeutic protein, and/or ii) a firstbiologically active nucleic acid molecule. In other embodiments, suchfirst and second compositions are sequentially administered (e.g.,systemically) to a subject such that the first therapeutic proteinand/or the biologically active nucleic acid molecule is/are expressed inthe subject (e.g., at a therapeutic level, for at least 5 or at least 50days, such that a disease or condition is treated or a physiologicaltrait is altered).

Work conducted during the development of embodiments of the presentdisclosure has shown that a single injection (e.g., intravenousinjection) of cationic liposomes, followed shortly thereafter byinjection (e.g., intravenous injection) of CpG-free vectors encoding atherapeutic protein produces circulating protein levels many times(e.g., 10-20 times higher) than the therapeutic serum level for theprotein for a prolonged period. Such administration also increasedcirculating neutrophil counts many fold weeks after the treatment.

Work conducted during the development of embodiments of the presentdisclosure (e.g., as shown in Example 1 below) has shown that a singleintravenous injection of cationic liposomes, followed two minutes laterby intravenous injection of CpG-free plasmid vectors encoding humangranulocyte-colony stimulating factor (hG-CSF) produces circulatinghG-CSF protein levels 10-20 times higher than the therapeutic serumhG-CSF level (greater than or equal to 100 pg/ml) for at least 63 days(see, FIG. 3). Such administration also increased circulating neutrophilcounts 10 fold, 3 weeks following intravenous injection into mice (FIG.4). In contrast, one systemic injection of cationic liposome-DNAcomplexes containing a similar, but CpG-containing) hG-CSF plasmidvector was unable to produce detectable (>20 pg/ml) hG-CSF proteinlevels even at day 3 after injection, and failed to increase neutrophilcounts at any point after injection (see, Tu et al., JBC, 275(39):30408-30416, 2000; herein incorporated by reference in itsentirety). Moreover, the approach presented in Example 1 that was usedto prolong expression at therapeutic levels of human G-CSF did notappear to cause significant toxicity in the mice.

Thus, the approach provided herein for expression in vivo overcomes thecritical limitation that has up to now precluded the successfultherapeutic application of systemic non-viral gene delivery. Namely, itsinability to express delivered genes at therapeutic levels for theextended periods generally required to produce important therapeutic orphysiological endpoints. As shown in Example 1, embodiments of themethods provided herein accomplish such long lasting expression of atherapeutic protein with non-viral vectors without having to incorporateviral genes into the vectors. This is important as other approachesrelied on the insertion of at least one viral gene plus the viral DNAsequence to which its protein product binds (the EBNA-1 gene togetherwith the EBV family of repeat sequences inserted into the DNA vector)has been required in order to overcome this transient gene expressionlimitation (see, Tu et al., above). Moreover, in addition to the highhG-CSF protein levels found after 63 days in Example 1 (FIG. 1), similarhigh levels of expression were measured and found on days 14, 21, 28,and 49 after injection, indicating that once achieved, these hightherapeutic levels are maintained longer term. Also, Example 2 shows, inFIG. 17, over 400 days of high levels of expression. This high level andlong term expression is significantly better than the mRNA approachprovided by MODERNA, which, as shown in FIG. 3 of U.S. Pat. No.8,754,062 for hG-CSF, only produced therapeutic levels of up to 4 daysafter a single IV injection.

In addition, the systems, methods, and compositions provided hereinprovide a versatile (e.g., non-viral) gene delivery and expressionplatform that can much more precisely control the duration of expressionof delivered genes at therapeutic levels. This ability to control theduration of expression of delivered genes addresses another up to nowcritical unmet need within the gene therapy field, the ability tocontrol the duration at which proteins are expressed at therapeuticlevels. Specifically, there is now a wide and expanding spectrum ofFDA-approved, recombinant, secreted human protein therapies. Differentapproved protein therapies must be present at therapeutic levels forvery different durations in order to both effectively and safely treatpatients. Recommended treatment durations of different protein therapiesvary from less than two weeks (hG-CSF) to the lifetime of the patient(factor IX). For example, recombinant human G-CSF protein, Neupogen, isgiven daily for only the first 10 days of each three-week chemotherapycycle. Serum hG-CSF levels return to baseline approximately 14 hoursafter each daily Neupogen dose. This 10 day treatment schedule is usedbecause its neutrophil increasing effect is indicated only during thisapproximately 10 day period of chemotherapy-induced neutropenia. G-CSFelevation from days 11 to 21 is generally not beneficial, as thepatient's own neutrophil producing capacity returns. Giving Neupogenbeyond day 10 can cause toxic, neutrophilia-related side effects. Incontrast, anti-TNF antibodies are routinely administered for months oryears, and factor IX replacement for the lifetime of the patient. Thus,different proteins must be produced at therapeutic levels for differentdurations, from less than two weeks to the lifetime of the patient.Therefore, a gene therapy approach that can control the duration of geneexpression at therapeutic levels it produces in patients achievestherapeutic endpoints while avoiding toxic side effects for a widespectrum of now FDA-approved, human therapeutic proteins. Providedherein are various technologies that can be employed to provide thiscontrol. Five exemplary approaches are described below.

First, in certain embodiments, a second expression cassette is insertedinto a single plasmid DNA vector or other vector. As shown in Example 1,in contrast to the single expression cassette hG-CSF plasmid vector thatwas used, which produces therapeutic hG-CSF levels for at least 63 days(FIG. 3), adding the second cassette limited therapeutic levels ofhG-CSF protein produced to less than two weeks in mice (FIG. 5). Ofnote, the second expression cassette which drives the luciferase gene,is also expressed at high, controllable levels in IV injected mice.

Second, as shown in Example 1, a series of different, CPG-freepromoter-enhancer combinations were generated in single cassette plasmidvectors that express hG-CSF at therapeutic levels for a range ofdifferent durations following a single IV injection in mice (FIG. 6). Ofnote, multi-expression cassette, single plasmid DNA vectors that containdifferent cassettes incorporating different promoter enhancercombinations are capable of expressing different therapeutic proteins atdifferent levels for different durations from a single DNA vector. Thisallows a single DNA vector to express multiple different therapeuticproteins (e.g., one, two, three, four, five, six or more therapeuticproteins). Each individual protein is then expressed for the requiredduration at its appropriate therapeutic level. Such an approach is oneway to overcome the prohibitive costs now incurred by combining two ormore recombinant protein therapies in a single patient.

Third, as described in Example 1, it was shown that co-injecting nowFDA-approved drugs, singly or in selected combinations with the cationicliposomes can selectively either increase or decrease the level/durationof expression of the gene subsequently delivered by sequential IVinjection in mice (FIGS. 7 and 8).

Fourth, as demonstrated in Example 1, varying the cationic liposomesize, as well as the lipid composition can also control the level andduration of expression of genes delivered by sequential cationicliposome then DNA injection (FIG. 11).

Fifth, as demonstrated in example 14, the addition of neutral lipidstogether with dexamethasone and dexamethasone palmitate can increase theduration of gene expression (FIGS. 26 and 29). In contrast, theadministration of neutral lipid alone can decrease the duration of geneexpression (FIGS. 14 and 15).

In addition, some literature also describes that matrix attachmentregions (MAR) should be incorporated into DNA vectors in order toproduce prolonged expression following their IV injection (Argyros etal., J Mol Med (2011) 89:515-529, herein incorporated by reference inits entirety). In contrast, work conducted during development ofembodiments of the present disclosure indicate that the presence of suchMAR elements do not increase, and in some vectors decrease the durationof gene expression produced by IV, sequential injection of cationicliposomes followed by CPG-free plasmid DNA (FIG. 9).

In certain embodiments, the present disclosure employs polycationicstructures (e.g., empty cationic liposomes, empty cationic micelles, orempty cationic emulsions) not containing vector DNA, which areadministered to a subject prior to vector administration. In certainembodiments, the polycationic structures are cationic lipids and/or areprovided as an emulsion. The present disclosure is not limited to thecationic lipids employed, which can be composed, in some embodiments, ofone or more of the following: DDAB, dimethyldioctadecyl ammoniumbromide; DPTAP (1,2-dipalmitoyl 3-trimethylammonium propane); DHA;prostaglandin, N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammoniummethyl sulfate; 1,2-diacyl-3-trimethylammonium-propanes, (including butnot limited to, dioleoyl (DOTAP), dimyristoyl, dipalmitoyl, disearoyl);1,2-diacyl-3-dimethylammonium-propanes, (including but not limited to,dioleoyl, dimyristoyl, dipalmitoyl, disearoyl) DOTMA,N-[1-[2,3-bis(oleoyloxy)]propyl]-N,N,N-trimethylammoniu-m chloride;DOGS, dioctadecylamidoglycylspermine; DC-cholesterol,3.beta.-[N-(N′,N′-dimethylaminoethane)carbamoyl]cholesterol; DOSPA,2,3-dioleoyloxy-N-(2(sperminecarboxamido)-ethyl)-N,N-dimethyl-1-propanami-niumtrifluoroacetate; 1,2-diacyl-sn-glycero-3-ethylphosphocholines(including but not limited to dioleoyl (DOEPC), dilauroyl, dimyristoyl,dipalmitoyl, distearoyl, palmitoyl-oleoyl); beta-alanyl cholesterol;CTAB, cetyl trimethyl ammonium bromide; diC14-amidine,N-t-butyl-N′-tetradecyl-3-tetradecylaminopropionamidine; 14Dea2,O,O′-ditetradecanolyl-N-(trimethylammonioacetyl) diethanolaminechloride; DOSPER, 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide;N,N,N′,N′-tetramethyl-N,N′-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butan-ediammoniumiodide; 1-[2-acyloxy)ethyl]2-alkyl (alkenyl)-3-(2-hydroxyethyl-)imidazolinium chloride derivatives such as1-[2-(9(Z)-octadecenoyloxy)eth-yl]-2-(8(Z)-heptadecenyl-3-(2-hydroxyethyl)imidazoliniumchloride (DOTIM),1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2-hydroxyethyl)imidazoliniumchloride (DPTIM);1-[2-tetradecanoyloxy)ethyl]-2-tridecyl-3-(2-hydroxyeth-yl)imidazoliumchloride (DMTIM) (e.g., as described in Solodin et al. (1995) Biochem.43:13537-13544, herein incorporated by reference); 2,3-dialkyloxypropylquaternary ammonium compound derivates, containing a hydroxyalkyl moietyon the quaternary amine, such as 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI); 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE); 1,2-dioleyloxypropyl-3-dimethyl-hydroxypropylammonium bromide (DORIE-HP),1,2-dioleyloxypropyl-3-dimethyl-hydroxybutyl ammonium bromide(DORIE-HB); 1,2-dioleyloxypropyl-3-dimethyl-hydroxypentyl ammoniumbromide (DORIE-HPe); 1,2-dimyristyloxypropyl-3-dimethyl-hydroxylethylammonium bromide (DMRIE);1,2-dipalmityloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DPRIE); 1,2-disteryloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide(DSRIE) (e.g., as described in Felgner et al. (1994) J. Biol. Chem.269:2550-2561, herein incorporated by reference in its entirety). Manyof the above-mentioned lipids are available commercially from, e.g.,Avanti Polar Lipids, Inc.; Sigma Chemical Co.; Molecular Probes, Inc.;Northern Lipids, Inc.; Roche Molecular Biochemicals; and Promega Corp.

In certain embodiments, the present disclosure employs CpG-reduced orCpG-free expression vectors. An initial sequence that contains CpGdinucleotides (e.g., wild-type version of human G-CSF), may be modifiedto remove CpG dinucleotides by altering the nucleic acid sequence. FIG.2 shows a CpG-free version of human G-CSF, with sequences that have beenchanged to removed CpGs underlined. Such CpG di-nucleotides can besuitably reduced or eliminated not just in a coding sequence, but alsoin the non-coding sequences, including, e.g., 5′ and 3′ untranslatedregions (UTRs), promoter, enhancer, polyA, ITRs, introns, and any othersequences present in the nucleic acid molecule or vector. CpGdi-nucleotides may be located within a codon triplet for a selectedamino acid. There are five amino acids (serine, proline, threonine,alanine, and arginine) which have one or more codon triplets thatcontain a CpG di-nucleotide. All five of these amino acids havealternative codons not containing a CpG di-nucleotide that can bechanged to, to avoid the CpG but still code for the same amino acid asshown in Table 1 below. Therefore, the CpG di-nucleotides allocatedwithin a codon triplet for a selected amino acid may be changed to acodon triplet for the same amino acid lacking a CpG di-nucleotide.

TABLE 1 DNA Codons DNA Codons Amino Acid Containing CpG Lacking CpGSerine (Ser or S) TCG TCT, TCC, TCA, AGT, AGC Proline (Pro or P) CCGCCT, CCC, CCA, Threonine (Thr or T) ACG ACA, ACT, ACC Alanine (Ala or A)GCG GCT, GCC, GCA Arginine (Arg or R) CGT, CGC, AGA, AGG CGA, CGGIn addition, within the coding region, the interface between tripletsshould be taken into consideration. For example, if an amino acidtriplet ends in a C-nucleotide which is then followed by an amino acidtriplet which can start only with a G-nucleotide (e.g., Valine, Glycine,Glutamic Acid, Alanine, Aspartic Acid), then the triplet for the firstamino acid triplet is changed to one which does not end in aC-nucleotide. Methods for making CpG sequences are shown, for example,in U.S. Pat. No. 7,244,609, which is herein incorporated by reference. Acommercial service provided by INVIVOGEN is also available to produceCpG free (or reduced) nucleic acid sequences and vectors.

Provided below in Table 2 are exemplary promoters and enhancers that maybe used in the vectors described herein. Such promoters, and otherpromoters known in the art, may be used alone or with any of theenhancers, or enhancers, known in the art. Additionally, when multipleproteins or biologically active nucleic acid molecules (e.g., two,three, four, or more) are expressed from the same vector, the same ordifferent promoters may be used in conjunction with the subject nucleicacid sequence.

TABLE 2 Promoter Enhancer CMV human CMV EF1α mouse CMV Ferritin(Heavy/Light) Chain SV40 GRP94 Ubc U1 AP1 UbC hr3 Beta Actin IE2 PGK1IE6 GRP78 E2-RS CAG MEF2 SV40 C/EBP TRE HNF-1

The present disclosure is not limited by the type of therapeuticproteins that is expressed. In certain embodiments, the therapeuticprotein comprises an antibody or antibody fragments (e.g., F(ab) orF(ab′)2). In other embodiments, the therapeutic protein is selected fromthe group consisting of an anti-inflammatory protein, coagulationprotein, anti-cancer protein, anti-sepsis protein, etc.

EXAMPLES Example 1 In Vivo Protein Expression Using Sequential Injectionof Cationic Liposomes Followed by CPG-Free Expression Vectors

This example describes various work using in vivo protein expressionusing sequential injection of cationic liposomes followed closely intime by CPG-free expression vectors.

Methods Liposome Preparation.

Pure DOTAP lipid as a lyophilized powder was purchased from Avanti polarlipids. Pure DOTAP cationic liposomes were prepared by re-suspending thelyophilized powder in a solution of 5% dextrose in water at a lipidconcentration of 20 millimolar. The solution was then vortexed for 15minutes to form multi-lamellar vesicles (MLV), mean particle size 350nm, as measured by laser light scattering. Small uni-lamellar vesicles(SUV), mean particle size 75 nm, were then formed from MLV by sonicationin a bath sonicator.

Plasmid Construction.

General schematics for the vectors employed are provided in FIG. 1. Ingeneral, a CpG free DNA plasmid vector is typically composed of thefollowing elements: enhancer/promoter/5′UTR of either mCMV/EF1/I126 (851bp) or hCMV/hCMV/HTLV (873 bp), linked to a gene of interest (such ash-GCSF (615 bp) or soLux (1653 bp)), minimal polyA (63 bp), MARs derivedfrom either βGlobin (434 bp), 21q21 (1055 bp) or IFN (820 bp) and an R6KOri/Kan^(r) (Kanamycin antibiotic resistant) expression cassette (1206bp). R6KOri/Kan^(r) DNA was designed as a base vector containing threeendonuclease restriction enzyme sites, DraIII, EcoRI and NheI. It wasassembled from gBlock four DNA fragments (IDT, IA) using the GibsonAssembly technique (NEB, MA). For MAR containing plasmids, a βGlobin MARwas inserted into the base vector at DraIII-EcoRI sites. The CpG-freenucleic acid sequence for h-GCSF is shown in FIG. 2.

The expression cassette was constructed using the puc19 plasmid backboneby sequentially inserting each DNA element between EcoRI and XbaI.Enhancer/Promoter elements containing 5′ EcoRI and NheI sites wereligated to the 5′UTR, gene, pA or pA-MAR, as well as pucl9 at EcoRI,EcoRV, BstEII, BglII and XbaI sites, respectively. The expressioncassette was then digested with EcoRI-XbaI and inserted into the basevector at EcoRI-NheI, producing an expression plasmid containingrestriction sites that can be used to insert a second expressioncassette insertion. Dual (Luc- and GCSF) cassette expression plasmidswere then constructed by inserting the hG-CSF expression cassette intothe base vector at EcoRI-NheI. The second, Luc expression cassette wassubsequently inserted into the G-CSF expression plasmid at EcoRI-NheI,producing a dual cassette, Luc and GCSF containing, single plasmidvector.

Plasmid Purification.

Endotoxin-free plasmids were purified on 5′Prime Endo-free Maxi columnsas follows. Briefly, 200 ml of bacteria containing the plasmid are grownovernight at 37 C and then collected. Bacterial cells are lysed per themanufacturer's protocol. Endotoxin is removed using an EndoFree filterCS. Isopropanol is added to the lysate and then loaded onto a column.After successive washes, the column is centrifuged and air-dried for 10min to ensure residual ethanol is removed. DNA is then eluted from thecolumn with 1 ml of Lactated Ringers.

Mice.

21 g female, CD-1 mice were purchased from Charles River. Housing, careand all procedures were performed according to IACUC approvedguidelines.

Sequential Injection of Cationic Liposomes, then Plasmid DNA in Mice.

Three to five mice were injected per group. Each mouse received a singleIV injection of cationic liposomes (MLV or SUV), followed two minuteslater by a single IV injection of a CPG-free, plasmid DNA vector.

Obtaining and then Analyzing Mouse Serum for Human G-CSF Levels.

Each mouse was anesthetized and then bled via the submandibular vein.Serum was then isolated from whole blood and human G-CSF levels measuredin pg/ml, as performed strictly according to the manufacturer'sspecifications, using an R and D systems human G-CSF ELISA.

Obtaining and then Analyzing Mouse Tissue for Luciferase Activity.

Lung was homogenized with 500 ul of 1× Lysis buffer (Promega, Wis.). Thehomogenate was centrifuged at 3000×g at 4 C for 10 min. and thesupernatant collected. Luciferase activity was assayed using 20 μl ofsupernatant and 100 μl of Luciferase reagent for 10 seconds using aGloMax® Luminometer (Promega, Wis.).

Results/Description Serum Human G-CSF Levels Produced in Mice bySequential, IV Cationic Liposome Injection Followed by IV DNA VectorInjection

Five mice were injected per group. Each mouse received a single IVinjection of 800 nmoles of pure DOTAP cationic liposomes (MLV or SUV),followed two minutes later by a single IV injection of 80 μg of anmCMV-EF1-hGCSF, an hCMV-hCMV-hGCSF or an mCMV-EF1-luciferase, CPG-free,plasmid DNA vector. Serum hG-CSF levels were assessed beginning at dayseven after IV injection, and at seven-day intervals thereafter.

As shown in FIG. 3, all three DNA vectors containing the hG-CSF geneproduced supra-therapeutic hG-CSF levels (≥100 pg/ml is required toincrease neutrophil levels), at day seven after injection. Thereafter,hG-CSF levels rose progressively until day 21, and then remained stableuntil day 63, the last time point analyzed. In contrast, hG-CSF levelsproduced by identical, IV sequential injection of the EF1-luciferase DNAvector were undetectable throughout the course of the experiment.

WBC and Absolute Neutrophil Counts, 21 Days after Sequential, IVCationic Liposome then DNA Vector Injection

Whole blood was collected from groups of 4 mice at day 21 followingsequential, IV injection of DOTAP MLV, followed two minutes later by asingle IV injection of either an EF1-hGCSF or an EF-1luciferase-containing, CPG-free plasmid DNA vector. Blood from eachmouse was then analyzed, in a blinded fashion, for total WBC, as well asabsolute neutrophil counts by the University of California Davisveterinary diagnostics laboratory.

As shown in FIG. 4, one sequential IV injection of DOTAP cationicliposomes followed by IV injection of EF1-hG-CSF DNA increased absoluteneutrophil counts approximately 10 fold and total WBC approximately 4fold, 21 days following injection when compared to mock-injected controlmice receiving sequential injection of an EF1-luciferase, plasmid DNAvector. These results document that the hG-CSF gene encoded proteinproduct was fully functional in treated mice. Taken together, thehigh-level increases in absolute neutrophil counts produced by theEF1-hG-CSF DNA vector, coupled with the 10 to 15 fold above therapeutichG-CSF protein levels produced at day 21 (see FIG. 3) demonstrate that asingle, sequential IV injection of a cationic liposomes followed by aCPG-free DNA vector can produce prolonged therapeutic effects of a nowFDA-approved recombinant human protein therapy.

Serum Human G-CSF Levels Produced in Mice by Sequential IV Injection ofEither Single or Dual Cassette, hG-CSF Single Plasmid Vectors

Sera were collected from groups of four mice at either day 6 or 14following sequential, IV injection of 800 nmoles of pure DOTAP MLVcationic liposomes, followed two minutes later by IV injection of 40 μgeither an EF1-luciferase--EF1-hGCSF (2 expression cassette) or anEF1-hGCSF (1 expression cassette), CPG-free, single plasmid DNA vector.

As shown in FIG. 5, the single as well as dual cassette DNA vectorscontaining the hG-CSF gene each produced supra-therapeutic serum hG-CSFlevels (≥100 pg/ml required to increase neutrophil counts) at day sixafter injection, 951 and 423 pg/ml, respectively. The single cassettevector produced even higher therapeutic levels, 1941 pg/ml, at day 14.In contrast, hG-CSF levels produced at day 14 by the dual cassette,single plasmid DNA vector had fallen to a sub-therapeutic level (93pg/ml), as hG-CSF protein levels below 100 pg/ml are sub-therapeutic.Thus, adding a second expression cassette can control the duration ofexpression of the gene contained in the first cassette.

Serum hG-CSF Levels in Mice, 21 Days after IV Injection of CationicLiposomes, then DNA Containing Different Promoter-Enhancer CombinationsLinked to the hG-CSF Gene

Sera were collected from groups of four mice, 21 days followingsequential, IV injection of 800 nmoles of pure DOTAP MLV cationicliposomes, followed two minutes later by IV injection of 60 μg of thehG-CSF gene, linked to one of the following enhancer-promotercombinations, mCMV-EF1, hCMV-hCMV, hCMV-hferritin light chain,hCMV-hferritin heavy chain, hCMV-glucose-regulated protein 78 ormCMV-hferritin light chain, each in a CPG-free, single cassette, DNAvector.

As shown in FIG. 6, a range of supra-therapeutic, hG-CSF serum levelswere produced at day 21 by the mCMV-EF-1 (2120 pg/ml), hCMV-hCMV (1516pg/ml), hCMV-FerL (699 pg/ml), hCMV-Grp78 (343 pg/ml) and mCMV-FerL (303pg/ml)-driven DNA vectors, each linked to the hG-CSF gene. In contrast,the hCMV-FerH-hG-CSF DNA vector (52 pg/ml) produced a sub-therapeutichG-CSF level. Taken together, these results reveal that changing thepromoter-enhancer combination can produce a range of different hG-CSFprotein levels, from more than 20 fold above therapeutic to subtherapeutic, 21 days after a single injection. (hG-CSF proteinlevels≥100 pg/ml are required to increase neutrophil counts).

Mouse Lung Luciferase Levels, 7 Days after Sequential IV Injection ofCationic Liposomes, then Single Cassette, EF1-Luciferase DNA Alone orTogether with a Drug

Lungs were collected from groups of four mice, 7 days followingsequential, IV injection of 800 nmoles of pure DOTAP MLV cationicliposomes alone, or containing 2 mg/kg of L-arginine, 0.01 mg/kg ofcolchicine or 1 mg/kg of dexamethasone. In each case, cationic liposomeinjection was followed two minutes later by IV injection of 40 μg of anmCMV-EF1-luciferase, CPG-free, single cassette, DNA vector.

As shown in FIG. 7, when compared to mice receiving sequential injectionof DOTAP MLV alone (control), mice receiving either colchicine ordexamethasone together with the liposomes showed higher luciferaseactivity in the lung. In contrast, mice receiving L-arginine togetherwith liposomes failed to increase gene expression levels. Thus,co-injecting selected drugs together with the liposomes can increase thelevel and duration of expression of genes delivered by sequentialcationic liposome then DNA injection.

Mouse Lung Luciferase Levels, 7 Days after Sequential IV Injection ofCationic Liposomes, then Dual Cassette, EF1-hG-CSF-EF1-Luciferase DNAAlone or with Drug(s)

Lungs were collected from groups of four mice, 7 days followingsequential, IV injection of 800 nmoles of pure DOTAP MLV cationicliposomes alone, or containing 2 mg/kg of L-arginine, 1 mg/kg ofdexamethasone, 0.02 mg/kg of sildenafil, 0.1 mg/kg of valproic acid or 2mg/kg of L-arginine plus 0.02 mg/kg of sildenafil (VIAGRA). In eachcase, cationic liposome injection was followed two minutes later by IVinjection of 40 μg of EF1-luciferase--EF1-hGCSF, a 2 expressioncassette, CPG-free single plasmid DNA vector.

As shown in FIG. 8, when compared to mice receiving sequential injectionof DOTAP MLV alone (control), mice receiving dexamethasone, valproicacid or sildenafil alone, or L-arginine plus sildenafil together withthe liposomes showed higher luciferase activity in the lung. Incontrast, mice receiving either L-arginine or valproic acid togetherwith liposomes were either lower than or comparable to controls. Thus,depending on the drug co-injected, expression levels of the deliveredgene can be increased or reduced.

Mouse Lung Luciferase Levels, 10 Days after Sequential IV Injection ofCationic Liposomes, then a DNA Vector Containing One of a Series ofDifferent Promoter-Enhancer Combinations, Each Either with or withoutMARs and all Linked to the Luciferase Gene

Lungs were collected from groups of three mice, 10 days followingsequential, IV injection of 800 nmoles of pure DOTAP MLV cationicliposomes, followed two minutes later by IV injection of 40 μg of theluciferase gene, linked to one of the following enhancer-promotercombinations: hCMV-hCMV, hCMV-human ferritin heavy chain, hCMV-CBOX(human Carboxypeptidase B1), mCMV-hCMV, mCMV-CBOX and mCMV-EF1, eachlinked to the luciferase gene in a CPG-free, single cassette, DNAvector.

FIG. 9 shows that DNA vectors lacking MARs, and containing the hCMVenhancer linked to the hCMV, ferritin heavy chain or CBOX promotersproduced higher lung luciferase levels than the corresponding vectorscontaining MAR elements. In contrast, DNA vectors containing both MARsand the mCMV enhancer linked to the hCMV, EF1 or CBOX promoters failedto produce lung luciferase levels as high as the corresponding vectorslacking MAR elements. Thus, CPG-free DNA vectors lacking MARs canproduce more durable expression than MAR-containing vectors.

Mouse Lung Luciferase Levels, 1 or 5 Days after IV Injection of PEI:EF-1Luc DNA Complexes or Sequential IV Injection of Cationic Liposomes, thenthe Identical EF-1 Luc DNA

Lungs were collected from groups of three mice, 1 or 5 days following IVinjection of either 12.5 μg of CPG-free EF-1-Luc DNA vector complexed to22 kDa linear PEI at a 1:4 N:P ratio, or sequential, IV injection of 900nmoles of pure DOTAP MLV cationic liposomes, followed two minutes laterby IV injection of 40 μg of the same EF-1-Luc DNA vector.

FIG. 10 shows lung luciferase levels were consistently higher in thePEI:DNA injected mice than in the mice injected sequentially withcationic liposomes then DNA at day one following injection. However,lung luciferase levels had fallen approximately 100 fold in the PEI:DNAinjected mice by day five. In direct contrast, lung luciferase levelsfrom the sequentially IV injected mice had risen by day 5 afterinjection. Luciferase levels were up to 150 fold higher insequentially-injected mice than those present in PEI:DNA injected micealso sacrificed at day 5. Thus, sequential cationic liposome then DNAinjection produces higher levels of gene expression at later time pointswhen compared to the same CPG free DNA injected as a PEI:DNA complex.

Mouse Lung Luciferase Levels, 1 Day after Sequential IV Injection of Oneof Seven Different Cationic Liposome Formulations, then Single Cassette,EF1-Luciferase DNA.

Lungs were collected from groups of four mice, 1 day followingsequential, IV injection of 800 nmoles of pure DOTAP MLV, pure DOTAPSUV, DOTAP:cholesterol 2:1 MLV, DOTAP:diolelyl phosphatidylcholine(DOPC) 1:1 MLV, DSTAP MLV, ethyl DSPC MLV or DOTAP:DOBAQ 1:1 MLVcationic liposomes. In each case, cationic liposome injection wasfollowed two minutes later by IV injection of 80 μg of anmCMV-EF1-luciferase, CPG-free, single cassette, DNA vector.

As shown in FIG. 11, when compared to lung luciferase levels in micereceiving sequential IV injection of pure DOTAP MLV, mice receivingDOTAP SUV, DOTAP:chol or DOTAP:DOPC cationic liposome formulationsproduced gene expression levels approximating DOTAP MLV or higher. Incontrast, mice receiving DSTAP, ethyl DSPC or DOTAP:DOBAQ MLV producedeither very low or nearly undetectable lung luciferase levels. ThatDOTAP SUV produced gene expression levels approximating DOTAP MLV wasunexpected because DOTAP MLV produces more than 1700 fold higher levelsof gene expression than DOTAP SUV when injected as cationic liposome:DNAcomplexes (see, Nature Biotechnology, 15:167-173; 1996, hereinincorporated by reference in its entirety).

Mouse Spleen Luciferase Levels, 1 Day after IV Injection of PEI:EF-1 LucDNA Complexes Alone or Mixed with One of Four Different Drugs.

Spleens were collected from groups of three mice, 1 day following IVinjection of 12.5 μg of CPG-free EF-1-Luc DNA vector complexed to 22 kDalinear PEI at a 1:4 N:P ratio. Mice received an intraperitonealinjection of one ml of 5% DMSO either alone, or containing 200 μg ofamlexanox, 1 mg of chloroquine, 200 μg of SAHA or 300 μg of tofacitinibper mouse, two hours prior to receiving IV PEI:DNA complexes.

FIG. 12 shows pre-injection of the anti-inflammatory agents amlexanox,chloroquine or SAHA prior to injecting CPG-free DNA increased geneexpression levels, whereas tofacitinib failed to increase geneexpression. Amlexanox in particular, a selective inhibitor of theTBK1-induced interferon activation pathway, increased gene expressionlevels. Thus, pre-injection of selected anti-inflammatory agents mayfurther increase the effectiveness of CPG-free DNA for gene therapy.

Mouse Lung Luciferase Levels, 1 or 7 Days after Sequential IV Injectionof Cationic Liposomes, then One of a Series of Dual Cassette,EF-1-Luc-hG-CSF DNA Vectors

Lungs were collected from groups of four mice, 1 or 7 days followingsequential, IV injection of 800 nmoles of pure DOTAP MLV cationicliposomes followed two minutes later by 40 μg of EF1-Luc-EF1-hGCSF,EF1-Luc-hCMV-hCMV-hGCSF, EF1-Luc-hCMV-hCBOX-hGCSF,EF1-Luc-hCMV-hREG1-hGCSF or EF1-Luc-mCMV-hCBOX-hGCSF 2 expressioncassette, CPG-free single plasmid DNA vector.

FIG. 13 shows that when compared to mice receiving sequential injectionof EF1-Luc-EF1-hGCSF dual cassette vector, containing the EF1 promoterin each cassette (control), mice receiving each of the other four dualcassette DNA vectors containing the EF1 promoter in one cassette andanother promoter in the second cassette showed consistently higherluciferase activity in the lung at both day one and seven afterinjection. Dual cassette vectors containing a different promoter in eachcassette produced lung luciferase levels up to tenfold or more higher ateach time point than produced by the dual cassette vector containing theEF-1 promoter in both cassettes. Thus, using different promoter elementsin different cassettes of multi-cassette vectors can significantlyincrease the level and duration of gene expression they produce.

Serum Human G-CSF Levels Produced in Mice, 1 Day after Sequential, IVCationic Liposome Injection, with or without Co-Injection of NeutralLiposomes, Followed by IV Injection of a Dual Cassette, Single PlasmidVector.

Neutral MLV liposomes were prepared from Phospholipon 90H, Lipoid GmbH.The fatty acid content of this product is 15% palmitic acid, 85% stearicacid. Liposomes were prepared either by drying down the lipids inorganic solvent on a rotary evaporator, then re-suspending the driedlipid film in a solution of 5% dextrose in water at a lipidconcentration of 50 millimolar or by hydrating the lipid as a dry powderin 5% w/v dextrose. Both were prepared at 60 degrees C. The solution wasthen vortexed for 15 minutes to form MLV. Sera were collected fromgroups of four mice at day 1 following sequential, IV injection ofbuffer alone (control), 1000 nmoles of pure DOTAP SUV cationicliposomes, alone or co-injected with 1000 nmol of neutral MLV, followedtwo minutes later by IV injection of 120 μg an EF1-luciferase--EF1-hGCSF(2 expression cassette), CPG-free, single plasmid DNA vector or 1400nmoles of pure DOTAP SUV cationic liposomes, alone or co-injected with1000 nmol of neutral MLV, followed two minutes later by IV injection of100 μg of EF1-luciferase--EF1-hGCSF DNA.

As shown in FIG. 14, serum hG-CSF levels produced one day aftersequentially co-injecting pure DOTAP SUV together with neutral MLV thenEF1-luciferase--EF1-hGCSF DNA were increased from 3 to 600 fold whencompared to sequential injection of DOTAP SUV without neutral MLV. Thus,co-injecting neutral liposomes together with cationic liposomes cansignificantly increase peak levels of gene expression produced. Inaddition, co-injecting neutral liposomes appears to eliminate thevariation in gene expression levels produced by sequentially injectingdifferent ratios of cationic liposomes to DNA without co-injectingneutral liposomes.

Serum Human G-CSF Levels Produced in Mice, 1 or 7 Days after Sequential,IV Cationic Liposome Co-Injection with Neutral Liposomes, Followed by IVInjection of a Dual Cassette Plasmid Vector

Sera were collected from groups of four mice at day 7 followingsequential, IV injection of 1000 nmoles pure DOTAP SUV cationicliposomes co-injected with 1000 nmol of neutral MLV, followed twominutes later by IV injection of 120 μg of CPG-free,EF1-luciferase--EF1-hGCSF DNA or 1000 nmoles of pure DOTAP SUV cationicliposomes co-injected with 1400 nmol of neutral MLV, followed twominutes later by IV injection of 100 μg of EF1-luciferase--EF1-hGCSFDNA. Sera were also collected from groups of four mice at day 1following sequential, IV injection of buffer only (control), 800 nmolesof pure DOTAP SUV cationic liposomes co-injected with 1000, 750, 500,250 or 100 nmol of neutral MLV respectively, followed two minutes laterby IV injection of 90 μg of EF1-luciferase--EF1-hGCSF.

As shown in FIG. 15, serum hG-CSF levels produced by co-injecting pureDOTAP SUV together with neutral MLV dropped by approximately 100 foldcompared to the hG-CSF levels produced in the same mice one day afterinjection, (See FIG. 14 for hG-CSF levels produced at day 1 followinginjection of these same mice). Thus, co-injecting neutral liposomes withcationic liposomes can strongly alter both peak as well as longer-termexpression of delivered genes. In addition, the ratio of neutral tocationic liposomes co-injected determines the extent to whichco-injected neutral liposomes increase the expression of sequentiallydelivered genes.

Mouse Serum hG-CSF Levels, 7 Day after Sequential IV Injection ofDifferent Cationic Liposome Formulations, then Single Cassette, CPG-FreeEF1-hG-CSF DNA.

Sera were collected from groups of four mice, 7 days followingsequential, IV injection of either 800 or 1000 nmoles of pure DOTAP SUV,or MLV cationic liposomes. Injection of each of the three cationicliposome formulations was followed two minutes later by IV injection ofeither 100 or 120 μg of an mCMV-EF1-h-G-CSF, CPG-free, single cassette,DNA vector. 0.1 μm extruded cationic liposomes were prepared from MLV bysequential extrusion through sized polycarbonate membranes under highargon gas pressure in a Lipex extrusion device.

As shown in FIG. 16, and in part depending on the ratio of nmolescationic liposomes to μg DNA ratio injected, pure DOTAP SUV and 0.1 μmextruded cationic liposomes produced extended, high-level expression ofhG-CSF as efficiently as that produced by MLV cationic liposomes.Therefore, SUV as well as 0.1 μm extruded (oligolamellar) cationicliposomes are as effective as MLV when used for sequential cationicliposome then CPG-free DNA injection.

Example 2 Long-Term Expression

In this Example, five mice per group were sequentially injected with 800nmol of either DOTAP MLV or SUV followed by 90 ug of a CPG-free plasmidvector containing an EF1- or hCMV-driven hG-CSF cDNA. Serum levels ofhuman G-CSF protein were assessed at 7- or 14-day intervals for thesubsequent 428 days following injection. Obtaining and analyzing mouseserum for human G-CSF levels was performed as follows. Each mouse wasanesthetized and bled via submandibular vein. Serum was isolated fromwhole blood using serum separator tubes from BD. Human G-CSF levels weremeasured in pg/ml via an ELISA performed strictly according to themanufacturer's specifications, using an R&D systems human G-CSF ELISA.The results are shown in FIG. 17, and show that supra-therapeutic levelsof human G-CSF protein were produced in fully immune-competent mice forat least 428 days after receiving a single IV injection of DOTAP SUVliposomes then an EF1-huG-CSF plasmid DNA vector.

Example 3 Protein Expression in Rats

In this Example, 250 gm Sprague-Dawley female rats #22 and 23 weresequentially injected with 6000 nmol of DOTAP SUV then 300 ug of aCPG-free plasmid vector containing an EF1-driven hG-CSF DNA vector.Serum levels of human G-CSF protein, WBC and absolute neutrophil counts(ANC) were assessed at 7-day intervals following injection. Serum ALTand AST levels were assessed at day 1 only. All were assessed by the UCDavis Comparative Pathology lab. As shown in FIG. 18, and Table 3 below,supra-therapeutic levels of hG-CSF protein, as well as significantlyelevated WBC and ANC, were produced in EF1-huG-CSF injected rats for atleast 22 days following a single IV injection. ALT and AST measured atday 1 after injection were comparable to background control levels inun-injected rats.

TABLE 3 WBC Day 1 Day 8 Day 15 Day 22 ALT Day 1 #22 6.56 27.36 23.6827.20 Ctrl 4.57 #22 30.30 Ctrl 12-67 #23 11.04 15.74 12.90 20.08 SEM0.27 #23 29.40 ANC Day 1 Day 8 Day 15 Day 22 AST Day 1 #22 4.99 17.3112.32 15.69 Ctrl 1.39 #22 89.80 Ctrl 14-113 #23 5.07 8.07 6.68 10.57 SEM0.13 #23 74.00

Example 4 DPTAP Liposomes

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 900 nmoles of DOTAP, DMTAP, or DPTAP(1,2-dipalmitoyl 3-trimethylammonium propane) SUV liposomes followed twominutes later by a single IV injection of 70 ug of an EF-1 plasmid DNAvector encoding hG-CSF. Serum levels of hG-CSF were determined by ELISA24 hours following injection. FIG. 19 shows that HuG-CSF protein waspresent in serum from mice treated with DOTAP or DPTAP but not DMTAP.These data indicate that multiple cationic lipids can mediatetransfection in vivo, and that level of protein production can becontrolled by selection of the lipid carrier.

Example 5 Toxicity Resolves within 48 Hours

In this Example, three mice were injected per group. Mice were purchasedfrom Charles River Labs. Each mouse received a single IV injection of1000 nmoles, 1200 nmoles, or 1400 nmoles of DOTAP SUV liposomes asindicated, followed two minutes later by a single IV injection of 100μg, 120 μg, or 140 μg of a CPG-free EF-1 driven plasmid DNA vectorencoding luciferase. Serum was collected at 24 hrs or 48 hrs afterinjection. ALT and AST measurements were assayed at the UC DavisComparative Pathology lab. As shown in FIG. 20, at 24 hours followingsequential injection, serum levels of ALT and AST were elevated from twoto five fold in all lipid then DNA groups. At 48 hours, serum ALT andAST levels returned to control (background) levels (shown by themock-injected group). These data indicate that toxicity as measured byALT/AST is acute (present within 24 hrs of injection) and transient(gone by 48 hrs).

Example 6 Liposomes with DexP

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 900 nmoles of either pure DOTAP SUV liposomesor each mouse received a single IV injection of 900 nmoles of eitherpure DOTAP SUV liposomes or liposomes containing indicated mole % s ofdexamethasone covalently linked to palmitate (DexP) incorporated intothe liposome bilayer. Incorporation of 5% cholesteryl palmitate (CholP)into the liposome bilayer served as a control. This was followed twominutes later by a single IV injection of 90 ug of plasmid DNA encodinghG-CSF. Serum levels of hG-CSF were determined by ELISA 24 hoursfollowing injection and ALT measurements were assayed at the UC DavisComparative Pathology lab. As shown in FIG. 21, at 24 hours followingsequential injection, toxicity as measured by ALT levels is 2-3 foldhigher than seen in animals that were mock injected with lactatedringer's solution only (ALT Control). Incorporation of 2.5%dexamethasone palmitate (DexP) into the liposome bilayer produced a dualeffect of increasing peak expression of hG-CSF as well as reducing ALTlevels to within 1.5 fold of background (normal) levels at 24 hours.

Example 7 DexP Reduces Toxicity and Increases Expression

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 900 nmoles of either pure DOTAP SUV liposomesor liposomes containing 2.5 mole % of dexamethasone covalently linked topalmitate (DexP) incorporated into the liposome bilayer of DOTAPliposomes. This was followed two minutes later by a single IV injectionof either 130 ug (high) or 40 μg (low) of an EF-1 driven plasmid DNAvector encoding hG-CSF. Two groups were treated two hours prior to IVinjection with an IP injection of 1 umole of dexamethasone palmitate.Serum levels of hG-CSF were determined by ELISA 24 hours followinginjection and ALT measurements were assayed at 24 hours by the UC DavisComparative Pathology lab. As shown in FIG. 22, sequential injection of130 ug of DNA produced significantly higher hG-CSF protein levels than40 ug of DNA 24 hours later. Inclusion of 2.5 mole % dexamethasonepalmitate in the liposomes at either DNA dose further increased hG-CSFprotein levels. In addition, incorporation of dexamethasone palmitate inliposomes reduced ALT levels to within 1.5 fold of background (normal)levels, even at the much higher DNA dose.

Example 8 Pre and Post Dex Injection

In this Example, mice were pre-injected IP with 15 mg/kg of Tofacitinibor 40 mg/kg of Dexamethasone, followed 2 hours later by sequential IVinjections of 900 nmol DOTAP SUV, then 70 ug of a CPG-free, EF-1 drivenhG-CSF plasmid vector. Another IP injection of Tofacitinib orDexamethasone was administered 2 hours after injection of DNA. As shownin FIG. 23, administration of dexamethasone prior to, as well asfollowing, sequential cationic liposome then DNA injection bothsignificantly increased HuG-CSF protein levels while concurrentlyreducing toxicity within 1.5 fold of background (normal) levels. Incontrast, pre- and post-injection of the immunosuppressive agentTofacitinib did neither.

Example 9 Lipid to DNA Ratio

In this Example, three mice per group were given IV injections of 900,1000, 1200, or 1500 nmols of DOTAP SUV liposomes containing 2.5 mole %of dexamethasone covalently linked to palmitate (DexP) incorporated intothe liposome bilayer of DOTAP liposomes, suspended in Lactated Ringer's(LR) to a final volume of 100 uL per injection, followed 2 minutes laterby 40, 60, or 75 ug of a CPG-free, EF-1 driven, hG-CSF plasmid vector at100 per injection. Mock-injected mice received LR only without lipid orDNA. Serum levels of hG-CSF protein and ALT were assayed 24 hours later.As shown in FIG. 24, hG-CSF protein and ALT levels of mice sequentiallyinjected with DOTAP SUV lipid to plasmid DNA (nmole lipid:mg DNA) ratioslower than 26:1 produced significantly higher hG-CSF protein levelswhile preventing toxicity, as documented by producing ALT levels eitherwithin 1.5 fold of or equal to background (normal) levels in controlmice that received neither lipid nor DNA injection.

Example 10 Rituximab Expression

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 1000 nmoles of either pure DOTAP cationicliposomes or liposomes containing 2.5 mole % of dexamethasone covalentlylinked to palmitate (DexP) incorporated into the liposome bilayer ofDOTAP liposomes. This was followed two minutes later by a single IVinjection of 100 ug of a dual cassette, single plasmid DNA vectorencoding the Rituximab heavy and light chains (see constructs in FIGS.36, 40, and 41). Serum Rituximab levels were determined by ELISA 24hours following injection and then at 7-day intervals. Mice were bledand serum isolated as for G-CSF. Rituximab levels were measured using anImmunoguide ELISA obtained from Eagle Biosciences, and performedaccording to instructions. As shown in FIG. 25, IP dexamethasonepretreatment plus incorporation of 2.5 mole % dexamethasone palmitate inDOTAP liposomes increases serum Rituximab levels by more than five foldfor at least three weeks after injection.

Example 11 Rituximab Expression

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 1000 nmoles of either pure DOTAP cationicliposomes or liposomes containing 2.5 mole % of dexamethasone covalentlylinked to palmitate (DexP) incorporated into the liposome bilayer ofDOTAP liposomes. This was followed two minutes later by a single IVinjection of 100 ug of an EF-1-driven, dual cassette, single plasmid DNAvector encoding Rituximab (see constructs in FIGS. 36, 40, and 41). Onegroup was treated two hours prior to IV injection with an IP injectionof 40 mg/kg dexamethasone (Dex) and 1000 nmoles the neutral lipid (NL),DMPC. Serum Rituximab levels injected mice were determined by ELISA 24hours following injection and at 7-day intervals thereafter. As shown inFIG. 26, all mice produced significant levels of serum Rituximab proteinfor at least 12 weeks following one injection. Mice receiving thecombination of Dex, DexP and NL produced significantly higher serumRituximab levels over time. These data show that a single sequentialinjection of a dual cassette Rituximab plasmid DNA vector can producesignificant levels of serum Rituximab protein in animals for greaterthan 90 days.

Example 12 Dual Cassette, Single Plasmid Rituximab Expression

In this Example, Raji cells (1 million/sample) were incubated with mouseserum samples or recombinant Rituximab (50 ng/ml) for 1 hr at 4 C, inFACS binding buffer containing EDTA and 0.5% BSA. Following washes,samples were incubated with fluorescently labeled secondary antibody(anti-human IgG-PE) for 30 min, washed and analyzed using an Accuri flowcytometer. Between 3500-5000 events were recorded for each sample. Theexperiment was repeated twice with similar results.

FIG. 27 shows a FACS plots display fluorescence intensity for fourexperimental conditions. The upper panels show samples containing mouseserum from control (HuG-CSF) DNA plasmid vector injected mice orsecondary antibody alone, which display low, background levels offluorescence in the PE channel (˜300). The lower two panels showrecombinant Rituximab protein (left panel) and mouse serum followingRituximab plasmid DNA vector administration (right panel). Both samplesshow fluorescence intensities over 10 fold higher than the background asshown in Table 4 below, demonstrating that Rituximab present in themouse serum binds to target CD20-expressing human Raji B cells to anextent similar to recombinant Rituximab protein. Thus, the Rituximabpresent in mouse serum six weeks after injection of a dual cassette,single plasmid Rituximab DNA vector binds CD20+ target human B cells ina fully functional manner.

TABLE 4 Mean Fluorescence Sample designation Intensity Secondaryantibody 331.86 alone Mouse Serum (injected 279.32 with control plasmid)Recombinant Rituximab 5781.87 (50 ug/ml) Mouse Serum (injected 3532.40with anti-CD20 plasmid)

Example 13 Functional Rituximab is Expressed

In this Example, Raji cells (5×10⁴ cells/well) were plated in 96 wellplates using RPMI+10% FBS medium. Next day cells were incubated withRituximab (1, 10 ug/ml) or mouse serum samples (20 μl/well) for 1 hourat room temperature. Twenty ul of pooled normal human plasma (InnovativeResearch) was then added to all wells (except the Rituximab controlcondition) and the plates incubated for another 12h at 37 C. Cellviability was measured using the Promega Cell titer glo reagentaccording to the manufacturer's instructions. In FIG. 28, values areshown as percentage change from the control conditions in which serumfrom mice injected with a huG-CSF DNA plasmid DNA vector was used.Individual mouse sera were tested from five different mouse groups thatreceived a single sequential injection of a dual cassette, singleplasmid Rituximab DNA vector from 8 to 78 days prior to serumcollection.

Results of this Example are shown in FIG. 28. Sera from mice previouslyinjected with a dual cassette, single plasmid Rituximab DNA vector wereanalyzed first by ELISA to quantitate serum Rituximab concentrations.Adding these Rituximab-containing sera in a cell lysis assay then showedthat they lyse CD-20+ human Raji B cells in a manner comparable torecombinant Rituximab (Invivogen). Moreover, functional serum Rituximabprotein with documented lytic activity was isolated from animals acrossfive separate injection experiments over a eleven-week period,demonstrating its reproducible lytic efficacy over time.

Example 14 Enhanced Expression of Rituximab

In this Example, two, 250 gm Sprague-Dawley female rats per group werewere first pre-injected with 40 mg/kg dexamethasone, then sequentiallyinjected with 4400 nmol of DOTAP SUV liposomes containing 2.5 mole % ofdexamethasone covalently linked to palmitate (DexP) incorporated intothe liposome bilayer, with or without 4400 nmol of neutral DMPC lipid,then 360 μg of a dual cassette, single plasmid DNA vector containing anEF1-driven Rituximab cDNA (see constructs in FIGS. 36, 40, and 41).Serum levels of human Rituximab protein were assessed at 7-day intervalsfollowing injection. FIG. 29 shows significantly higher levels of serumRituximab protein were produced in rats also receiving neutral lipid forat least 15 days following a single IV injection.

Example 15 Codon-Optimized Rituximab Expression

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 1000 nmoles of liposomes containing 2.5 mole %of dexamethasone covalently linked to palmitate (DexP) incorporated intothe liposome bilayer of DOTAP liposomes. This was followed two minuteslater by a single IV injection of 100 ug of dual cassette, singleplasmid EF-1-driven DNA vector encoding Rituximab (see constructs inFIGS. 36, 42, and 43). Numbered plasmids (in FIG. 30) werecodon-optimized versions of the original, CpG-free but not codonoptimized Rituximab DNA sequence. Serum levels of Rituximab weredetermined by ELISA 24 hours following injection. FIG. 30 shows that at24 hours following sequential injection, codon-optimized DMA vector 6produced significantly higher levels of serum Rituximab protein thannon-codon optimized rituximab DNA vectors.

Example 16 Codon-Optimized Rituximab Expression

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of DOTAP cationic liposomes (900 nmoles or 1050nmoles as indicated) containing 2.5 mole % of dexamethasone covalentlylinked to palmitate (DexP) incorporated into the liposome bilayer. Thiswas followed two minutes later by a single IV injection of 75 ug of adual cassette, codon-optimized single plasmid DNA encoding Rituximab(see constructs in FIGS. 36, 42, and 43). Both groups were treated twohours prior to IV injection with an IP injection of 40 mg/kgdexamethasone. Serum levels of Rituximab were determined by ELISA 24hours following injection and at 7-day intervals thereafter. FIG. 31shows that one sequential IV injection of codon-optimized dual cassette,single plasmid Rituximab DNA vectors produces extended serum Rituximablevels for at least the next 60 days. Serum Rituximab levels rise overtime after a single IV sequential injection.

Example 17 Valproic Acid and Theophylline Increase Protein Expression

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 1050 nmoles of liposomes containing 2.5 mole %of dexamethasone covalently linked to palmitate (DexP) incorporated intothe liposome bilayer of DOTAP liposomes. This was followed two minuteslater by a single IV injection of 75 ug of dual cassette plasmid DNAencoding Rituximab. All groups were treated two hours prior to IVinjection with an IP injection of 40 mg/kg dexamethasone. Whereindicated in FIG. 32, animals were also pretreated by IP injection of 15mg/kg Valproic Acid (VPA), 2 mg/kg VPA, 30 mg/kg Theophylline (Theo) or15 mg/kg Theo. Serum Rituximab levels were determined by ELISA 24 hoursfollowing injection. FIG. 32 shows serum Rituximab levels produced weresignificantly increased by pre-treatment with the drugs Valproic Acid orTheophylline, thus providing a framework for further enhancing proteinlevels without altering the dose of lipid or DNA.

Example 18 Dual or Single Cassette Rituximab Expression

In this Example, mice were pre-injected with 40 mg/kg Dexamethasone IP.Two hours later, they were sequentially injected with either 1050 or1500 nmols of DOTAP SUV liposomes containing 2.5 mole % of dexamethasonecovalently linked to palmitate (DexP) incorporated into the liposomebilayer, followed by 60 μg or 75 μg of plasmid DNA. Plasmid DNAconstructs injected were either codon optimized, double-cassette, singleplasmid DNA vectors (see constructs in FIGS. 36, 42, and 43) or codonoptimized single-cassette plasmids (see constructs in FIGS. 36, 37, and39) containing Rituximab heavy and light chain sequences separated by a2A self-cleaving peptide DNA sequence. Serum Rituximab levels weredetermined by ELISA 24 hours following injection. FIG. 33 shows that at24 hours following sequential injection, mice that received 2A peptidecontaining single cassette vectors encoding Rituximab produced serumlevels approaching 400 ng/ml, approximately one-third the level producedby the dual cassette vector. Thus, significant Rituximab serum levelscan be produced by either dual- or single-cassette, 2Apeptide-containing DNA vectors.

Example 19 Lipid to DNA Ratio and Rituximab Expression

In this Example, three mice per group were given IV injections of 900,1050, 1200, 1500 or 1650 nmols of DOTAP SUV liposomes containing 2.5mole % of dexamethasone covalently linked to palmitate (DexP)incorporated into the liposome bilayer, suspended in Lactated Ringer's(LR) to a final volume of 100 uL per injection, followed 2 minutes laterby 75 ug of a CPG-free, dual cassette, single plasmid Rituximab vectorat 100 uL per injection. Mock-injected mice received LR only withoutlipid or DNA. Serum levels of Rituximab protein and ALT were assayed 24hrs later. FIG. 34 shows Rituximab protein and ALT levels of micesequentially injected with DOTAP SUV lipid to plasmid DNA (nmolelipid:mg DNA) ratios lower than 15:1 produced significantly higherRituximab protein levels while producing serum ALT levels within 1.5fold of background (normal) ALT levels in control mice that receivedneither lipid nor DNA injection.

Example 20 Factor IX Expression with Valproic Acid or Theophylline

In this Example, three mice were injected per group. Each mouse receiveda single IV injection of 1500 nmoles of DOTAP SUV liposomes containing2.5 mole % of dexamethasone covalently linked to palmitate (DexP)incorporated into the liposome bilayer. This was followed two minuteslater by a single IV injection of 60 ug of a codon optimized,EF-1-driven single cassette DNA vector encoding the human factor IX cDNA(see constructs in FIGS. 38 and 44). All groups were treated two hoursprior to IV injection with an IP injection of 40 mg/kg dexamethasone.Where indicated, animals were also pretreated by IP injection of 2 mg/kgValproic Acid (VPA) or 30 mg/kg Theophylline (Theo). Serum human factorIX levels were determined by ELISA 24 hours following injection. Eachmouse was bled as for G-CSF. Blood was collected into tubes containingPotassium EDTA or Sodium Citrate to prevent coagulation and centrifugedto obtain plasma. An AssayPro ELISA specific to human Factor IX was usedaccording to manufacturer's instructions to measure Factor IXexpression. FIG. 35 shows serum human factor IX levels produced at 24hrs were significantly higher in mice receiving the human factor IX DNAvector plus pre-treatment with either Valproic Acid or Theophylline.

Example 21 Size Determination of Liposomes

In this Example, the sizes of various liposomes were determined. Inparticular, the liposomes in Table 5 were prepared in 5% w/w glucose,and the size was determined using quasi elastic laser light scattering.The Z-Average particle size of these DOTAP liposomes is shown in Table5.

TABLE 5 Liposome Type Z-Average Particle Size (nm) DOTAP MultilamellarLiposomes (MLV) 339 DOTAP 0.1 micron Extruded MLV 146 DOTAP SonicatedLiposomes (SUV) 74

All publications and patents mentioned in the present application areherein incorporated by reference. Various modification and variation ofthe described methods and compositions of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the invention. Although the invention has been described inconnection with specific preferred embodiments, it should be understoodthat the invention as claimed should not be unduly limited to suchspecific embodiments. Indeed, various modifications of the describedmodes for carrying out the invention that are obvious to those skilledin the relevant fields are intended to be within the scope of thefollowing claims.

We claim:
 1. A system comprising: a) a first composition comprising afirst amount of polycationic structures liposomes, wherein said firstcomposition is free, or essentially free, of nucleic acid molecules; andb) a second composition comprises non-viral expression vectors, whereinsaid non-viral expression vectors are CpG-free or CpG-reduced, whereineach of said non-viral expression vectors comprises a first nucleic acidsequence encoding: i) a first therapeutic protein, and/or ii) a firstbiologically active nucleic acid molecule; and at least one of thefollowing: i) wherein the ratio of said first amount of saidpolycationic structures to said non-viral expression vectors is 5:1 to25:1; ii) wherein 2.0% to 6.0% of said first composition comprisesdexamethasone or dexamethasone palmitate; iii) wherein said firstcomposition further comprises neutral lipid; and iv) wherein saidpolycationic structures comprise empty liposomes, and wherein said emptyliposomes present in said first composition have a z-average diameter ofabout 20-85 nm.
 2. The system of claim 1, wherein said wherein saidpolycationic structures comprise empty liposomes, and wherein said emptyliposomes present in said first composition have a z-average diameter ofabout 20-85 nm.
 3. The system of claim 1, wherein said non-viralexpression vectors each further comprise an regulating nucleic acidsequence, wherein said regulating nucleic acid sequence reduces theduration of expression of said first nucleic acid sequence that wouldoccur in the absence of said regulating nucleic acid sequence.
 4. Thesystem of claim 3, wherein said regulating nucleic acid sequence isselected from the group consisting of: a promoter, an enhancer, a secondnucleic acid sequence encoding a second protein, and/or a secondbiologically active nucleic acid molecule,
 5. The system of claim 1,wherein said first amount of polycationic structures in said firstcomposition comprises a mixture of cationic lipid and neutral lipid thatreduces the expression of said first therapeutic protein and/or firstbiologically active nucleic acid molecule compared to such expressionwhen only said cationic lipid is employed in said method.
 6. The systemof claim 1, wherein said first nucleic acid sequence encodes saidtherapeutic protein, and wherein said therapeutic protein is selectedfrom the group consisting of: human G-CSF, Rituximab, and human FactorIX.
 7. The system of claim 1, wherein said non-viral expression vectorscomprise a first nucleic acid sequence encoding said first therapeuticprotein.
 8. The system of claim 1, wherein said non-viral expressionvectors comprise a first nucleic acid sequence encoding said firsttherapeutic protein, wherein said first therapeutic protein comprises amonoclonal antibody light chain.
 9. The system of claim 1, wherein saidnon-viral expression vectors comprise a first nucleic acid sequenceencoding said first therapeutic protein, wherein said first therapeuticprotein comprises a monoclonal antibody heavy chain.
 10. The system ofclaim 1, wherein said non-viral expression vectors comprise: i) a firstnucleic acid sequence encoding said first therapeutic protein, whereinsaid first therapeutic protein comprises a monoclonal antibody heavychain, and ii) a second nucleic acid sequence encoding a secondtherapeutic protein, wherein said second therapeutic protein comprises amonoclonal antibody light chain.
 11. The system of claim 1, wherein saidnon-viral expression vectors comprise a first nucleic acid sequenceencoding said first biologically active nucleic acid molecule.
 12. Thesystem of claim 1, wherein said non-viral expression vectors comprise afirst nucleic acid sequence encoding said first biologically activenucleic acid molecule, wherein said first biologically active nucleicacid molecule comprises a shRNA sequence.
 13. The system of claim 1,wherein said non-viral expression vectors comprise a first nucleic acidsequence encoding said first biologically active nucleic acid molecule,wherein said first biologically active nucleic acid molecule comprises amiRNA sequence.
 14. The system of claim 1, wherein said non-viralexpression vectors comprise a first nucleic acid sequence encoding saidfirst biologically active nucleic acid molecule, wherein said firstbiologically active nucleic acid molecule comprises an antisensesequence or ribozyme.
 15. The system of claim 1, wherein said non-viralexpression vectors comprise a first nucleic acid sequence encoding saidfirst biologically active nucleic acid molecule, wherein said firstbiologically active nucleic acid molecule comprises a CRISPR singleguide RNA sequence.
 16. The system of claim 1, wherein said at least oneof the following is i) wherein the ratio of said first amount of saidpolycationic structures to said non-viral expression vectors is 5:1 to25:1.
 17. The system of claim 1, wherein said at least one of thefollowing is ii) wherein 2.0% to 6.0% of said first compositioncomprises dexamethasone or dexamethasone palmitate.
 18. The system ofclaim 1, wherein said at least one of the following is iii) wherein saidfirst composition further comprises neutral lipid.
 19. The system ofclaim 1, wherein said at least one of the following is iv) wherein saidpolycationic structures comprise empty liposomes, and wherein said emptyliposomes present in said first composition have a z-average diameter ofabout 20-85 nm.